TW202216788A - Bispecific antibody binding to GPC3 and TfR - Google Patents

Abstract

The purpose of the present invention is to provide a bispecific antibody that binds to human GPC3 and human TfR, a fragment of the bispecific antibody, etc. The present invention pertains to: a bispecific antibody that binds to human GPC3 and human TfR; a fragment of the bispecific antibody; a nucleic acid containing a base sequence that encodes the bispecific antibody or the bispecific antibody fragment; a vector containing the nucleic acid; a transformant strain producing the bispecific antibody or the bispecific antibody fragment; a method for producing the bispecific antibody or the bispecific antibody fragment; a therapeutic agent and/or a diagnostic agent containing the bispecific antibody or the bispecific antibody fragment; a therapeutic method and/or a diagnostic method using the bispecific antibody or the bispecific antibody fragment; and a detection or assay reagent containing the bispecific antibody or the bispecific antibody fragment.

Description

Bispecific antibodies that bind to GPC3 and TfR

The present invention relates to a bispecific antibody that binds to human Glypican 3 (GPC3, Glypican 3) and human transferrin receptor (TfR, Transferrin Receptor), the bispecific antibody fragment, comprising encoding the Nucleic acid of base sequence of bispecific antibody or bispecific antibody fragment, vector containing said nucleic acid, transformant producing said bispecific antibody or said bispecific antibody fragment, said bispecific antibody or said bispecific antibody Production method of specific antibody fragment, therapeutic and/or diagnostic drug comprising the bispecific antibody or the bispecific antibody fragment, therapeutic method and/or diagnosis using the bispecific antibody or the bispecific antibody fragment Methods, and detection or assay reagents comprising the bispecific antibody or the bispecific antibody fragment.

Antibodies are glycoproteins present in the serum or tissue fluids of all mammals and mainly recognize foreign antigens in the organism. Antibodies function by neutralizing the function of foreign antigens, activation of the complement system, or binding to receptors (FcR) present on the cell surface, thereby activating FcR to express the phagocytic ability, antibody-dependent cytotoxicity, and mediator release ability of cells , and antigen presentation (antigen presentation) ability and other effector functions, involved in biological defense.

One molecule of antibody contains two identical light chains (L chains) and two identical heavy chains (H chains), and has two antigen-binding sites. The types and subtypes of antibodies are determined by the H chain, and each type and subtype have different inherent functions. Human antibodies exist in 5 different classes: IgG, IgA, IgM, IgD and IgE. IgG is further divided into subtypes of IgG1, IgG2, IgG3 and IgG4, and IgA is divided into subtypes of IgA1 and IgA2 (Non-Patent Document 1).

In recent years, in the study of highly functionalized antibodies, multivalent antibodies have attracted attention as molecules that exert new functions or activities that previous antibodies could not achieve. A polyvalent antibody is an antibody having a plurality of antigen-binding sites in one molecule. As an example of a multivalent antibody, the following report can be exemplified. First, by using the hybridoma method, H chain and L chain derived from two different antibodies are expressed in the same cell, thereby producing monovalent binding to different antibodies. Bivalent antibody of two antigens (Non-Patent Document 2). However, about 10 combinations of antibody H chain and L chain are produced by this method. Therefore, the production of a multivalent antibody with the desired combination of H and L chains is low, and it is also difficult to selectively isolate and purify such a multivalent antibody, so the yield of the desired antibody is reduced.

It has been reported that in order to overcome the above-mentioned problems, an attempt has been made to link a plurality of antigen-binding sites and express them in the form of a single polypeptide chain, thereby reducing the variation of the inter-chain combination and efficiently producing an antibody having a desired combination.

As an example, a multivalent antibody comprising a single-chain Fv (scFv) in which the antigen-binding sites of the H chain and the L chain are linked by one polypeptide is known (Non-Patent Document 3). Furthermore, it is reported that the CH1 domain of the H chain constant region of IgG1 or a partial fragment of this region and the L chain constant region, or a flexible linker (Gly-Gly-Gly-Gly-Ser) are used to link the two antigen-binding sites. Multivalent antibodies, etc. (Patent Document 1, Patent Document 2).

These previous multivalent antibodies suffer from the disadvantages of easy agglutination, low stability or low productivity. On the other hand, it was found that a multivalent antibody having a plurality of antigen-binding sites in a single H-chain polypeptide and the antibody heavy chain variable region bound via a linker comprising the amino acid sequence of an immunoglobulin region or a fragment thereof is not only stable The property is high and the productivity is also good (Patent Document 3).

Transferrin receptor (also referred to as TfR hereinafter) was identified as a cell membrane protein expressed on the surface of reticulocytes. TfR has a function of taking up iron bound to transferrin (hereinafter also referred to as Tf) into cells. Iron is an essential metal for maintaining cell homeostasis or cell proliferation. Therefore, TfR uptake is expressed in normal tissues with strong iron uptake, such as placental trophoblast cells, reticulocytes, and activated lymphocytes. In addition, it is known that it is highly expressed in various tumor cells that are actively proliferating (Non-Patent Document 4, Non-Patent Document 5). It is known that when TfR is cross-linked on the cell membrane, it promotes internalization by clathrin-dependent endophagy and is degraded. (Non-Patent Document 6, Non-Patent Document 7).

The expression level of TfR is lower in normal cells other than bone marrow cells, but higher in cancer cells with active proliferation. Therefore, TfR has been used as a molecular target for cancer therapy. As a TfR-targeting molecule, for example, a TfR-neutralizing antibody that exhibits strong efficacy in a mouse cancer-bearing model is known (Patent Document 4).

Glypican 3 (GPC3, SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, GTR2-2) is a GPI (Glycosylphospatidylinositol, glycosylated phosphatidylinositol) containing 580 amino acids ) anchored membrane proteins. GPC3 is known to be a cancer antigen that is highly expressed in hepatocellular carcinoma. In addition, it has also been reported in malignant melanoma, ovarian clear cell carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms tumor, testicular germ cell tumor, and liposarcoma. It is expected to be a target molecule or diagnostic marker for molecularly targeted anticancer drugs, and a cancer vaccine target (Non-Patent Document 8).

It is known that by using a molecule capable of binding to two membrane proteins, one antigen molecule can be used as a scaffold to internalize and decompose the other antigen molecule (Patent Document 5, Patent Document 6). Furthermore, it has been reported that when a peptide recognizing a cancer cell-specific membrane protein and a peptide recognizing TfR are linked by chemical conjugation or hybridoma fusion, under the condition of adding an iron chelator, it is reported that the cells expressing both antigens are displayed. Growth inhibitory activity (Patent Document 7). [Prior Art Literature] [Patent Literature]

Patent Document 1: Specification of US Patent Application Publication No. 2007/0071675 Patent Document 2: International Publication No. 2001/077342 Patent Document 3: International Publication No. 2009/131239 Patent Document 4: International Publication No. 2012/153707 Patent Document 5: International Publication No. 2013/138400 Patent Document 6: Specification of European Patent Application Publication No. 2808035 Patent Document 7: Specification of US Patent No. 5705614 [Non-patent literature]

Non-Patent Document 1: Charles A. J. et al., Immunobiology, 1997, Current Biology Ltd/Garland Publishing Inc. Non-patent document 2: Suresh et al., Methods Enzymol. 121, 210-228, 1986 Non-Patent Document 3: Kranz et al., J. Hematother. 5, 403-408, 1995 Non-patent document 4: Hamilton TA et al., PNAS USA 76 6406-10, 1979 Non-Patent Document 5: Larson SM et al., J Natl Cancer Inst. 64 41-53, 1980 Non-patent document 6: Liu AP et al., J Cell Biol. 191 1381-93, 2010 Non-patent document 7: Weissman AM et al., J Cell Biol. 102 951-8, 1986 Non-Patent Document 8: Mitchell Ho and Heungnam Kim, Eur J of Cancer. 2011 Nov;47(3):333-338

[Problems to be Solved by Invention]

The object of the present invention is to provide a bispecific antibody that binds to human GPC3 and human TfR, a fragment of the bispecific antibody, a nucleic acid comprising a base sequence encoding the bispecific antibody or the fragment of the bispecific antibody, and a nucleic acid containing the Nucleic acid vector, transformant for producing the bispecific antibody or bispecific antibody fragment, manufacturing method of the bispecific antibody or the bispecific antibody fragment, comprising the bispecific antibody or the bispecific antibody Fragments of therapeutic and/or diagnostic drugs, therapeutic and/or diagnostic methods using the bispecific antibody or bispecific antibody fragment, and assays or assays comprising the bispecific antibody or bispecific antibody fragment reagents. [Technical means to solve problems]

As means for solving the above-mentioned problems, the present invention provides a bispecific antibody or a fragment of the bispecific antibody that binds to human GPC3 and human TfR.

That is, the present invention relates to the following items. What is claimed is: 1. A bispecific antibody that binds to human glypican 3 (GPC3) and human transferrin receptor (TfR). 2. The bispecific antibody according to 1 above, which binds bivalently to human GPC3 and human TfR, respectively. 3. The bispecific antibody as described in 1 or 2 above, comprising (a1) an IgG part comprising the first antigen-binding domain and (b1) a second antigen-binding domain, the weight of the IgG part comprising the first antigen-binding domain; The C-terminus of the chain is linked directly or via a linker to the N-terminus of the second antigen-binding domain, and either the first antigen-binding domain or the second antigen-binding domain binds to human GPC3, and the other binds to human TfR . 4. The bispecific antibody according to 3 above, wherein the C-terminus of the heavy chain of the IgG portion comprising the first antigen-binding domain is directly linked to the N-terminus of the second antigen-binding domain. 5. The bispecific antibody according to 3 or 4 above, wherein the second antigen-binding domain is an antibody Fab. 6. The bispecific antibody according to 5 above, wherein the C-terminus of the heavy chain of the IgG portion comprising the first antigen-binding domain is linked directly or via a linker to the N-terminus of the heavy chain of the Fab of the antibody. 7. The bispecific antibody as described in 1 or 2 above, comprising (a2) an IgG portion comprising a second antigen-binding domain and (b2) a first antigen-binding domain, the C-terminus of the first antigen-binding domain being directly or via A linker is linked to the N-terminus of the heavy chain of the IgG portion comprising the second antigen binding domain, either of the first antigen binding domain and the second antigen binding domain binds to human GPC3 and the other binds to human TfR . 8. The bispecific antibody according to 7 above, wherein the C-terminus of the first antigen-binding domain is directly linked to the N-terminus of the heavy chain comprising the IgG portion of the second antigen-binding domain. 9. The bispecific antibody according to 7 or 8 above, wherein the first antigen-binding domain is an antibody Fab. 10. The bispecific antibody according to 9 above, wherein the C-terminus of the heavy chain of the Fab of the antibody is linked directly or via a linker to the N-terminus of the heavy chain of the IgG portion comprising the second antigen-binding domain. 11. The bispecific antibody according to any one of 3 to 10 above, wherein any one of the first antigen-binding domain and the second antigen-binding domain is an anti-human GPC3 IgG antibody Fab (anti-GPC3 Fab). 12. The bispecific antibody according to any one of 3 to 11 above, wherein any one of the first antigen-binding domain and the second antigen-binding domain is an anti-human TfR IgG antibody Fab (anti-TfR Fab). 13. The bispecific antibody according to any one of the above 3 to 12, wherein the first antigen-binding domain is an anti-GPC3 Fab, and the second antigen-binding domain is an anti-TfR Fab. 14. The bispecific antibody according to any one of 3 to 12 above, wherein the first antigen-binding domain is an anti-TfR Fab, and the second antigen-binding domain is an anti-GPC3 Fab. 15. The bispecific antibody as described in any one of the above 11 to 14, wherein the above-mentioned anti-GPC3 Fab is a Fab comprising a heavy chain variable region (VH) and a light chain variable region (VL), and the heavy chain variable region The region (VH) contains any one of complementarity determining regions (CDRs) 1 to 3 selected from the following (g1) to (g20), (g23) and (g25) to (g40), and the light chain variable region (VL ) contains CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 24 to 26, respectively, (g1) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 91 to 93, (g2) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 95 to 97, (g3) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 99 to 101, (g4) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 103 to 105, (g5) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 107 to 109, (g6) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 111 to 113, (g7) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 115 to 117, (g8) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 119 to 121, respectively, (g9) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 123 to 125, (g10) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 127 to 129, (g11) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 131 to 133, (g12) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 135 to 137, (g13) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 139 to 141, (g14) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 143 to 145, (g15) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 147 to 149, (g16) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 151 to 153, (g17) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 155 to 157, (g18) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 159 to 161, (g19) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 163 to 165, (g20) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 167 to 169, (g23) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 179 to 181, (g25) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 187 to 189, (g26) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 191 to 193, (g27) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 195 to 197, (g28) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 199 to 201, (g29) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 203 to 205, (g30) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 207 to 209, (g31) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 211 to 213, (g32) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 215 to 217, (g33) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 219 to 221, (g34) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 223 to 225, (g35) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 227 to 229, (g36) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 231 to 233, (g37) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 235 to 237, respectively, (g38) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 239 to 241, respectively, (g39) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 243 to 245, respectively, (g40) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 247 to 249, respectively. 16. The bispecific antibody as described in any one of the above 11 to 15, wherein the above-mentioned anti-GPC3 Fab is an anti-GPC3 Fab comprising a compound selected from the group consisting of SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, The VH of the amino acid sequence represented by any one of 242 and 246, and the Fab comprising the VL of the amino acid sequence represented by SEQ ID NO: 23. 17. The bispecific antibody according to any one of the above 12 to 16, wherein the anti-TfR Fab is a Fab comprising VH and VL, and the VH comprises CDR1~ 3. The VL contains CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 24 to 26, respectively. 18. The bispecific antibody according to any one of the above 12 to 16, wherein the anti-TfR Fab is a Fab comprising VH and VL, and the VH comprises CDR1~ 3. The VL contains CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 24 to 26, respectively. 19. The bispecific antibody according to any one of the above 12 to 17, wherein the anti-TfR Fab is a VH comprising the amino acid sequence represented by SEQ ID NO: 35 and a VH comprising the amino acid sequence represented by SEQ ID NO: 23. Fab of VL. 20. The bispecific antibody according to any one of the above 12 to 16 and 18, wherein the anti-TfR Fab is a VH comprising the amino acid sequence represented by SEQ ID NO: 40 and the amino acid comprising the amino acid sequence represented by SEQ ID NO: 23 Fab of VL of sequence. 21. The bispecific antibody according to any one of the above 3 to 20, wherein the IgG part comprising the first antigen-binding domain or the IgG part comprising the second antigen-binding domain comprises the amino acid represented by SEQ ID NO: 255 Sequence of the heavy chain constant region. 22. the bispecific antibody as described in any one of above-mentioned 1 to 6, 11 to 13, 15 to 17, 19 and 21, it comprises 2 heavy chains and 4 light chains, and these 2 heavy chains are respectively from N From the end, the VH including the amino acid sequence represented by any one of SEQ ID NOs: 90, 142, 126, 178, 198, 146, 218, 150, 202, and 226, and the amine represented by SEQ ID NO: 255 are included in this order. CH of the amino acid sequence, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and CH1 comprising the amino acid sequence represented by SEQ ID NO: 253; VL of the amino acid sequence and CL comprising the amino acid sequence represented by SEQ ID NO: 272. 23. The bispecific antibody as described in any one of above-mentioned 1, 2, 7 to 13, 15 to 17, 19 and 21, comprising 2 heavy chains and 4 light chains, the 2 heavy chains are respectively from N The terminus sequentially contains a sequence number selected from the group consisting of: VH of the amino acid sequence represented by any one of 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246, including SEQ ID NO: CH1 of the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and CH comprising the amino acid sequence represented by SEQ ID NO: 255; VL of the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272. 24. The bispecific antibody as described in any one of above-mentioned 1, 2, 7 to 13, 15, 16, 18, 20 and 21, comprising 2 heavy chains and 4 light chains, the 2 heavy chains are respectively From the N-terminus, it contains a sequence selected from the group consisting of SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, VH of the amino acid sequence represented by any one of 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246, including CH1 of the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 40, and CH comprising the amino acid sequence represented by SEQ ID NO: 255, the four light chains are each in sequence from the N-terminus VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 were included. 25. The bispecific antibody as described in the above 24, comprising 2 heavy chains and 4 light chains, the 2 heavy chains respectively from the N-terminus sequentially contain a sequence selected from SEQ ID NO: 94, 98, 102, 106, VH of the amino acid sequence represented by any one of 114, 130, 178 and 190, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 40, and SEQ ID NO: 40 CH of the amino acid sequence represented by 255, the four light chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 from the N-terminus, respectively. 26. The bispecific antibody as described in the above 24, comprising 2 heavy chains and 4 light chains, the 2 heavy chains respectively from the N-terminus sequentially contain a sequence selected from the group consisting of SEQ ID NOs: 94, 98, 102, 114, VH of the amino acid sequence represented by any one of 130, 166, 178, 186 and 190, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and comprising CH of the amino acid sequence represented by SEQ ID NO: 255, the four light chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 from the N-terminus, respectively . 27. The bispecific antibody according to any one of the above 1 to 26, which has cell proliferation inhibitory activity. 28. The bispecific antibody according to any one of the above 1 to 27, which does not inhibit the proliferation of cells that do not express GPC3 and express TfR, but inhibits the proliferation of cells that co-express GPC3 and TfR. 29. A bispecific antibody fragment, which is a bispecific antibody fragment of the bispecific antibody according to any one of the above 1 to 28. 30. A nucleic acid comprising a base sequence encoding the bispecific antibody according to any one of the above 1 to 28 or the bispecific antibody fragment according to the above 29. 31. A recombinant vector comprising the nucleic acid according to 30 above. 32. A transformed strain obtained by introducing the recombinant vector described in 31 above into a host cell. 33. A method for producing the bispecific antibody according to any one of the above 1 to 28 or the bispecific antibody fragment according to the above 29, wherein the transformed strain according to the above 32 is cultured in a medium, The bispecific antibody according to any one of the above 1 to 28 or the bispecific antibody fragment according to the above 29 is produced and accumulated in the culture, and the bispecific antibody or the bispecific antibody fragment is collected from the culture . 34. A therapeutic drug and/or diagnostic drug for a disease related to at least one of GPC3 and TfR, comprising the bispecific antibody as described in any one of 1 to 28 above or the bispecific antibody fragment as described in 29 above as an active ingredient. 35. The therapeutic and/or diagnostic drug according to 34 above, wherein the disease associated with at least one of GPC3 and TfR is cancer. 36. A method for treating and/or diagnosing a disease related to at least one of GPC3 and TfR, using the bispecific antibody as described in any one of 1 to 28 above or the bispecific antibody fragment as described in 29 above . 37. The method for treatment and/or diagnosis according to 36 above, wherein the disease associated with at least one of GPC3 and TfR is cancer. 38. The bispecific antibody according to any one of the above 1 to 28 or the bispecific antibody fragment according to the above 29, for use in the treatment and/or diagnosis of a disease associated with at least one of GPC3 and TfR. 39. The bispecific antibody or bispecific antibody fragment according to the above 38, wherein the disease associated with at least one of GPC3 and TfR is cancer. 40. Use of the bispecific antibody as described in any one of 1 to 28 above or the bispecific antibody fragment as described in 29 above, for the manufacture of a therapeutic drug for a disease associated with at least one of GPC3 and TfR and / or diagnostic drugs. 41. The use according to 40 above, wherein the disease associated with at least one of GPC3 and TfR is cancer. 42. A reagent for detecting or measuring at least one of GPC3 and TfR, comprising the bispecific antibody according to any one of 1 to 28 above or the bispecific antibody fragment according to 29 above. [Effect of invention]

According to the present invention, there can be provided a bispecific antibody that binds to human GPC3 and human TfR, a fragment of the bispecific antibody, a nucleic acid comprising a base sequence encoding the bispecific antibody or the fragment of the bispecific antibody, and a nucleic acid containing the bispecific antibody Nucleic acid vector, transformant for producing the bispecific antibody or bispecific antibody fragment, manufacturing method of the bispecific antibody or the bispecific antibody fragment, comprising the bispecific antibody or the bispecific antibody Fragments of therapeutic and/or diagnostic drugs, therapeutic and/or diagnostic methods using the bispecific antibody or bispecific antibody fragment, and assays or assays comprising the bispecific antibody or bispecific antibody fragment reagents.

The present invention relates to a bispecific antibody or the bispecific antibody fragment (hereinafter also referred to as the bispecific antibody or the bispecific antibody fragment).

GPC3 in the present invention is used in the same meaning as SGB, DGSX, MXR7, SDYS, Simpson-Golabi-Hehmel Syndrome, Type1 (SGBS), OCI-5, SGBS1 and GTR2-2.

GPC3 includes, for example, human GPC3 containing the amino acid sequence shown in GenBank Accession No. NP_004475. In addition, for example, the amino acid sequence that is included in the amino acid sequence shown in GenBank Accession No. NP_004475, GenBank Accession No. XP_005594665, or NP_057906 is deleted, substituted or added, and has the function of GPC3. the polypeptide.

The GPC3 of the present invention also includes: the amino acid sequence comprising GenBank Accession No. NP_004475, GenBank Accession No. XP_005594665 or GenBank Accession No. NP_057906 has preferably 70% or more, more preferably 80% or more, and more preferably 90% Polypeptides with amino acid sequences with homology of more than 95%, including amino acid sequences with homology of more than 95%, more than 96%, more than 97%, more than 98% and more than 99%, and have GPC3 functional peptides.

Polypeptides comprising amino acid sequences in which one or more amino acid residues are deleted, substituted, or added to the amino acid sequences shown in GenBank Accession No. NP_004475, GenBank Accession No. XP_005594665, or GenBank Accession No. NP_057906 can be identified by using the above-mentioned targeting. Mutation introduction method etc. are obtained by introducing site-directed mutagenesis into DNA encoding the amino acid sequence shown in GenBank Accession No. NP_004475, GenBank Accession No. XP_005594665, or GenBank Accession No. NP_057906, for example. The number of amino acids to be deleted, substituted or added is not particularly limited, preferably 1 to several tens, for example 1 to 20 amino acids, more preferably 1 to several, for example 1 to 5 amino acid.

Examples of the gene encoding GPC3 in the present invention include the human GPC3 gene including the nucleotide sequence shown in GenBank Accession No. NM_004484.

In addition, the gene encoding GPC3 of the present invention also includes, for example, a gene containing DNA encoding the following polypeptide, which is included in the base sequence shown in the GenBank Accession No. NM_004484. Deletion, substitution or addition of bases of one or more bases base sequence, and has the function of GPC3; a gene containing DNA encoding the following polypeptide, and the above-mentioned polypeptide comprises a base sequence with more than 60% homology, preferably 80%, with the base sequence shown in GenBank accession number NM_004484 The base sequence with the above homology, and more preferably the base sequence with more than 95% homology, and has the function of GPC3; and the gene containing the DNA encoding the following polypeptide, and the above-mentioned polypeptide includes and includes SEQ ID NO: GenBank registration DNA with the base sequence shown in No. NM_004484 hybridizes under stringent conditions and has the function of GPC3; etc.

As DNA hybridized under stringent conditions, it means using, for example, DNA containing the nucleotide sequence shown in GenBank Accession No. NM_004484 as a probe by colony hybridization, plaque hybridization, Southern blot hybridization, or DNA microarray. DNA capable of hybridization obtained by an array method or the like. Specifically, DNAs that can be identified by the following methods: DNAs immobilized with DNAs derived from colonies or plaques obtained by hybridization, or PCR products or oligoDNAs having the sequences Filters or slides, hybridized at 65°C in the presence of 0.7-1.0 mol/L sodium chloride [Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), After "DNA Cloning 1: Core Techniques, A Practical Approach", 2nd edition, Oxford University Press (Oxford University) (1995)], use 0.1 to 2 times the concentration of SSC solution (the composition of 1-fold concentration SSC solution contains 150 mmol/L sodium chloride and 15 mmol/L sodium citrate), at 65°C, wash the filter or glass slide. Examples of DNAs capable of hybridizing include DNAs having preferably 60% or more homology with the nucleotide sequence shown in GenBank Accession No. NM_004484, more preferably DNAs having 80% or more homology, and more Preferably, it is DNA with more than 95% homology.

Gene polytypes are often found in the base sequences of genes encoding proteins of eukaryotes. The gene encoding GPC3 of the present invention also includes such a gene that causes small-scale variation in the nucleotide sequence of the gene used in the present invention due to polytype.

Unless otherwise specified, the value of homology in the present invention may be a value calculated by using a homology search program known in the industry. For the base sequence, for example, BLAST [Journal of Molecular Biology (J. . Mol. Biol.)", 215, 403 (1990)], the values calculated by the default parameters, etc., for the amino acid sequence, BLAST2 ["Nucleic Acids Research", 25, 3389 (1997), Genome Research, 7, 649 (1997), http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] The value calculated by the default parameters Wait.

As default parameters, G (Cost to open gap) is 5 in the case of base sequences and 11 in the case of amino acid sequences; -E (Cost to open gap) extend gap)) is 2 in the case of the base sequence, 1 in the case of the amino acid sequence; -q (Penalty for nucleotide mismatch) is -3; -r (nucleus The reward for nucleotide match is 1; -e (expect value) is 10; -W (wordsize) is 11 residues in the case of base sequences, 3 residues in the case of amino acid sequences; -y [Dropoff(X) for blast extensions in bits] is 20 in the case of blastn, outside blastn 7 in the case of the program; -X (X dropoff value for gapped alignment in bits) is 15; and -Z (final dropoff value for gapped alignment in bits) Yuan) (final X dropoff value for gapped alignment in bits)) is 50 in the case of blastn and 25 in the case of programs other than blastn (http://www.ncbi.nlm.nih.gov/blast/html /blastcgihelp.html).

A polypeptide comprising a partial sequence of the amino acid sequence of GPC3 can be produced by a method known in the art. Transformants of the expression vector containing the partially deleted DNA were cultured. In addition, based on the polypeptide or DNA produced by the above-mentioned method, by the same method as the above-mentioned, for example, a partial sequence including the amino acid sequence shown in GenBank Accession No. NP_004475 can be obtained by deletion, substitution or addition of one or more pieces A polypeptide of amino acid sequence of amino acids. Furthermore, a polypeptide comprising a partial sequence of the amino acid sequence of GPC3, or a polypeptide comprising an amino acid sequence of one or more amino acids deleted, substituted or added to the partial sequence of the amino acid sequence of GPC3 can also be It is produced by chemical synthesis methods such as the perylmethoxycarbonyl (Fmoc) method and the tertiary butoxycarbonyl (tBoc) method.

As the extracellular region of GPC3 in the present invention, for example, the amino acid sequence of human GPC3 shown in GenBank Accession No. NP_004475 in NCBI (http://www.ncbi.nlm.nih.gov/), Using the well-known transmembrane region prediction program SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM ver.2 (http://www.cbs.dtu.dk/services/ Areas predicted by TMHMM-2.0/) or ExPASy Proteomics Server (http://Ca.expasy.org/). Specifically, the amino acid sequences shown in the 25th to 563rd positions of GenBank Accession No. NP_004475 can be exemplified.

The functions of GPC3 include, for example, promoting the binding of Wnt and Frizzled by forming a complex with Wnt, and promoting the proliferation or migration of cells expressing GPC3 by activating the Wnt pathway.

Examples of cells expressing GPC3 include hepatocellular carcinoma, malignant melanoma, ovarian clear cell carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms tumor, and testicular reproductive tumors. Cancer cells included in cell tumors, liposarcoma, etc.

It has been reported that there are differences in the expression of GPC3 in cancer cells of cancer patients, not limited to high expression, and some cancer patients have low to moderate expression (document: Yorita K et al., Liver Int. )”, 2011 Jan;31(1):120-31). The present inventors confirmed that the expression intensity of GPC3 in hepatocellular carcinoma varies from patient to patient according to the previous report by immunostaining. Therefore, as a function of the bispecific antibody or the bispecific antibody fragment, it is considered that it is important from a clinical point of view that GPC3 exhibits pharmacological effects even if it exhibits low to moderate expression.

The expression level of GPC3 can be judged based on the graph obtained by flow cytometry. The displayed position in the graph may vary depending on the voltage setting, sensitivity setting of the machine, the use of antibodies for colonization, staining conditions, and the use of dyes. The way of clustering is appropriately lined. In addition, the expression level of GPC3 can also be judged by immunostaining.

The degree of expression (low expression, moderate expression or high expression) of the marker of interest can be judged by comparison with the results of control cells assayed under the same conditions. As control cells, for example, HepG2 cells described in the section of Examples may be mentioned.

For example, with regard to the expression level of GPC3 in a cell cluster, using flow cytometry analysis, the expression level of GPC3 in the cell cluster is compared with the expression level of GPC3 in the HepG2 cell cluster (control cells with high GPC3 expression), and the observation When the performance is equal to that of the control cells, it can be judged as high performance, and when the performance is lower than that of the control cells, it can be judged as low performance or moderate performance.

TfR in the present invention is used in the same meaning as CD71, TFR1, TR, T9, p90, and IMD46. Examples of TfR include human TfR including the amino acid sequence shown in GenBank Accession No. NP_003225 or SEQ ID NO: 6, monkey TfR including the amino acid sequence shown in SEQ ID NO: 8, and the like. Also, for example, a polypeptide having the function of TfR is included in SEQ ID NO: 6 and GenBank Accession No. NP_003225, including an amino acid sequence in which one or more amino acids are deleted, substituted, or added.

The TfRs of the present invention also include those comprising: generally 70% or more, preferably 80% or more, and more preferably 90% or more homology to the amino acid sequence shown in SEQ ID NO: 6, GenBank Accession No. NP_003225 or SEQ ID NO: 8 Polypeptides with amino acid sequences of sex; including amino acid sequences with homology of more than 95%, more than 96%, more than 97%, more than 98% and more than 99%, and having the function of TfR.

A polypeptide comprising an amino acid sequence in which one or more amino acid residues are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 6, GenBank Accession No. NP_003225 or SEQ ID NO: 8 can be introduced by using site-directed mutagenesis [Molecular Colonization: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989), A Laboratory Manual for Molecular Biology, John Wiley International (1987-1997), Nucleic Acid Research, 10, 6487 (1982), "Proc. Natl. Acad. Sci. USA", 79, 6409 (1982), "Gene", 34, 315 (1985), "Nucleic Acid Research" ", 13, 4431 (1985), "Proceedings of the National Academy of Sciences of the United States of America", 82, 488 (1985)] etc., in, for example, the DNA encoding the amino acid sequence shown in SEQ ID NO: 6, GenBank accession number NP_003225 or SEQ ID NO: 8 obtained by introducing site-directed mutagenesis. The number of amino acids to be deleted, substituted or added is not particularly limited, preferably 1 to several tens, for example 1 to 20 amino acids, more preferably 1 to several, for example 1 to 5 amino acid.

Examples of the gene encoding TfR include the nucleotide sequence of human TfR shown in SEQ ID NO: 5 or GenBank Accession No. NM_003234, the nucleotide sequence of monkey TfR shown in SEQ ID NO: 7, and the like. In addition, the gene encoding TfR of the present invention also includes, for example, a gene containing a DNA encoding the following polypeptide, which is included in the deletion, substitution or addition of the base sequence shown in SEQ ID NO: 5, GenBank Accession No. NM_003234 or SEQ ID NO: 7 The base sequence of more than 1 base, and has the function of TfR; the gene containing the DNA encoding the following polypeptide, and the above-mentioned polypeptide contains the base sequence shown in SEQ ID NO: 5, GenBank Accession No. NM_003234 or SEQ ID NO: 7. It is preferably a base sequence with homology of 60% or more, more preferably a base sequence with homology of more than 80%, and more preferably a base sequence with homology of more than 95%, and has the function of TfR; And a gene containing a DNA encoding the following polypeptide, the polypeptide comprising a DNA hybridized under stringent conditions with the DNA comprising the nucleotide sequence shown in SEQ ID NO: 5, GenBank Accession No. NM_003234 or SEQ ID NO: 7, and has the function of TfR; etc. . The gene encoding TfR of the present invention also includes genes whose polytypes cause small-scale variation in the nucleotide sequence within the gene used in the present invention.

A polypeptide comprising a partial sequence of the amino acid sequence of TfR can be produced by a method known to the industry, for example, can be produced in the following manner: encoding the amino acid sequence shown in SEQ ID NO: 6, GenBank Accession No. NP_003225 or SEQ ID NO: 8 If a portion of the DNA is deleted, a transformant into which an expression vector containing the partially deleted DNA is introduced is cultured. In addition, based on the polypeptide or DNA produced by the above-mentioned method, it is possible to obtain, for example, a deletion in the partial sequence including the amino acid sequence shown in SEQ ID NO: 6, GenBank Accession No. NP_003225, or SEQ ID NO: 8 by the same method as above. A polypeptide that replaces or adds an amino acid sequence of more than one amino acid. Furthermore, a polypeptide comprising a partial sequence of the amino acid sequence of TfR, or a polypeptide comprising a partial sequence of the amino acid sequence of TfR deleted, substituted, or added to the amino acid sequence of one or more amino acids can also be It is produced by chemical synthesis methods such as the perylmethoxycarbonyl (Fmoc) method and the tertiary butoxycarbonyl (tBoc) method.

Examples of the extracellular region of TfR in the present invention include, for example, the amino acid sequence of human TfR shown in GenBank Accession No. NP_003225, using the known transmembrane region prediction program SOSUI (http://sosui.proteome. bio.tuat.ac.jp/sosuiframe0.html), TMHMM ver.2 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) or ExPASy Proteomics Server (http://Ca.expasy. org/) and other predicted areas, etc. Specifically, the amino acid sequence shown in SEQ ID NO: 2 or GenBank Accession No. NP_003225 from positions 89 to 760 may be mentioned.

As the function of TfR, the uptake of iron necessary for cell survival and proliferation can be exemplified. If the complex of iron and transferrin binds to TfR, it is engulfed into cells by endophagy. If the pH in the endosome is lowered, iron is freed from transferrin and transferred to the cytoplasm via DMT1 for cell proliferation or energy production. It is known that the complex of transferrin and TfR after iron release is not usually decomposed, but moves to the cell surface again (document: Yamashiro DJ et al., Cell 37 789-800, 1984).

Examples of cells expressing TfR include cells of various normal tissues represented by bone marrow and placental cells, or various cancer cells represented by colorectal cancer, head and neck cancer, brain tumor, hematopoietic tumor, liver cancer, and esophageal cancer, or HT29, HSC-2, RAMOS, K562, HepG2, OE21, T.Tn, U-937, HuH-7, HLE and other cancer cell lines.

The so-called antibody system refers to a gene derived from all or part of the gene encoding the variable region of the heavy chain and the constant region of the heavy chain, and the variable region of the light chain and the constant region of the light chain that constitute an immunoglobulin (referred to as "antibody gene"). ”) protein. Antibodies of the present invention also include antibodies or antibody fragments of any immunoglobulin type and subtype.

The so-called heavy chain (H chain) refers to the polypeptide with the larger molecular weight among the two polypeptides constituting the immunoglobulin molecule. The heavy chain determines the type and subtype of the antibody. IgA, IgD, IgE, IgG and IgM have α chain, δ chain, ε chain, γ chain and μ chain as heavy chains, respectively, and the constant regions of the heavy chains are characterized by different amino acid sequences. The so-called light chain (L chain) refers to the polypeptide with the smaller molecular weight among the two polypeptides constituting the immunoglobulin molecule. In the case of human antibodies, there are two types of light chains, κ chain and λ chain.

The variable region (V region) generally refers to a region rich in diversity existing in the amino acid sequence on the N-terminal side of immunoglobulins. The part other than the variable region forms a less diverse structure, so it is called the constant region (C region). The variable regions of the heavy and light chains associate to form an antigen-binding site, which determines the binding properties of an antibody to an antigen.

In the heavy chain of human antibodies, the variable region corresponds to No. 1 in the EU index of Kabat et al. (Kabat et al., "Sequences of proteins of immunological interest", 1991, 5th edition). The amino acid sequence from position 1 to position 117 corresponds to the amino acid sequence after position 118 in the constant region. In the light chain of a human antibody, the amino acid sequence from position 1 to position 107 in the numbering system proposed by Kabat et al. (Kabat numbering) corresponds to the variable region, and the amino acid sequence after position 108 corresponds to the variable region. constant region. Hereinafter, the heavy chain variable region or the light chain variable region is abbreviated as VH or VL.

An antigen-binding site refers to a site in an antibody that recognizes and binds to an antigen, and this site forms a three-dimensional structure complementary to an antigenic determinant (epitope). A strong intermolecular interaction occurs between the antigen-binding site and the epitope. The antigen binding site consists of VH and VL comprising at least three complementarity determining regions (CDRs). In the case of human antibodies, VH and VL each have 3 CDRs. These CDRs are referred to as CDR1, CDR2 and CDR3, respectively, in order from the N-terminal side.

Among the constant regions, the heavy chain constant region is designated as CH, and the light chain constant region is designated as CL. CHs are classified according to α chain, δ chain, ε chain, γ chain, and μ chain, which are the subtypes of heavy chains. CH is composed of a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain arranged in this order from the N-terminal side, and the CH2 domain and the CH3 domain are collectively referred to as an Fc region. On the other hand, CL is classified into two subtypes according to the Cλ chain and the Cκ chain.

The so-called monoclonal antibody system refers to an antibody secreted by an antibody-producing cell that maintains monoclonality and recognizes a single epitope. Monoclonal antibody molecules contain the same amino acid sequence (primary structure) and have the same structure as each other. The so-called polyclonal antibody system refers to a cluster of antibody molecules secreted by different cloned antibody-producing cells. The so-called oligoclonal antibody system refers to a cluster of antibody molecules mixed with a plurality of different monoclonal antibodies.

The so-called epitope refers to the structural site of the antigen recognized and bound by the antibody. Examples of epitopes include a single amino acid sequence recognized and bound by a monoclonal antibody, a three-dimensional structure including an amino acid sequence, an amino acid sequence bound to a sugar chain, and an amine including a sugar chain bound. The three-dimensional structure of the amino acid sequence, etc.

Examples of the monoclonal antibody in the present invention include an antibody produced by a fusion tumor, and a gene recombinant antibody produced by a transformant transformed with an expression vector containing the antibody gene.

Fusion tumors can be prepared, for example, by preparing an antigen, obtaining antigen-specific antibody-producing cells from an animal immunized with the antigen, and then fusing the antibody-producing cells with myeloma cells. A desired monoclonal antibody can be obtained by culturing the fusion tumor, or administering the fusion tumor to an animal to cancerize the fusion tumor ascites, and separating and purifying the culture medium or ascites. As the animal to be immunized with the antigen, any one capable of producing a fusion tumor can be used, and mice, rats, hamsters, and rabbits are suitably used. Furthermore, cells having antibody-producing ability can be obtained from such an immunized animal, immunized in vitro, and then fused with myeloma cells to produce a fusion tumor.

Examples of genetically recombinant antibodies in the present invention include recombinant mouse antibodies, recombinant rat antibodies, recombinant hamster antibodies, recombinant rabbit antibodies, humanized chimeric antibodies (also referred to as chimeric antibodies), humanized antibodies Antibodies (also known as CDR-grafted antibodies) and human antibodies are antibodies produced by genetic recombination technology. In the recombinant antibody, the variable region and constant region of the heavy chain and light chain derived from the animal can be determined according to the animal species and purpose of the subject. For example, when the animal species of the object is a human, the variable region can be derived from a human or a non-human animal such as a mouse, and the constant region and the linker can be derived from a human.

The so-called chimeric antibody system refers to an antibody comprising VH and VL of an antibody of an animal other than human (non-human animal) and CH and CL of a human antibody. As the non-human animal, any one capable of producing a fusion tumor, such as mouse, rat, hamster, and rabbit, can be used. Chimeric antibodies can be produced by obtaining cDNAs encoding VH and VL from non-human animal-derived fusion tumors that produce monoclonal antibodies, and inserting them into animal cells containing DNA encoding CH and CL of human antibodies, respectively, for expression. A vector for expression of a chimeric antibody is constructed by using the vector and introduced into animal cells for expression.

The so-called humanized antibody system refers to an antibody obtained by grafting the CDRs of VH and VL of a non-human animal antibody to the corresponding CDRs of VH and VL of a human antibody. The region other than the CDRs of VH and VL is called a framework region (hereinafter referred to as FR). A humanized antibody can be produced by constructing a cDNA encoding the amino acid sequence of the VH comprising the amino acid sequence of the CDRs of the VH of the non-human animal antibody and the amino acid sequence of the FR of the VH of any human antibody , and the cDNA encoding the amino acid sequence of the VL comprising the amino acid sequence of the CDR of the VL of the non-human animal antibody and the amino acid sequence of the VL of the FR of the VL of any human antibody, respectively inserted into the CH comprising the encoding human antibody A humanized antibody expression vector was constructed by using an animal cell expression vector containing DNA of CL and CL, and the vector was introduced into animal cells for expression.

Human antibodies originally refer to naturally occurring antibodies in the human body, and also include antibodies obtained from human antibody phage libraries produced by advanced genetic engineering, cell engineering, and developmental engineering techniques, and human antibody-producing transgenic animals.

Antibodies naturally occurring in the human body can be obtained, for example, by immortalizing human peripheral blood lymphocytes by infecting human peripheral blood lymphocytes with Epstein-Barr virus, etc., and performing colonization, thereby culturing the antibody-producing lymphocytes, and purifying the antibody from the culture supernatant. .

Human antibody phage library refers to a gene library obtained by inserting antibody genes prepared from human B cells into phage genes to express antibody fragments such as Fab and scFv on the surface of phage. Using the binding activity to the antigen-immobilized substrate as an index, phages expressing antibody fragments having the desired antigen-binding activity on the surface were recovered from the gene pool. The antibody fragment can be further transformed into a human antibody molecule comprising 2 complete H chains and 2 complete L chains by genetic engineering methods.

Transgenic animals for human antibody production refer to animals in which human antibody genes are embedded in cells. Specifically, for example, human antibody-producing gene-transfected mice can be produced by introducing human antibody genes into mouse ES cells, transplanting the ES cells into early mouse embryos, and allowing them to develop into individuals. Human antibodies derived from human antibody-producing transgenic animals can be prepared by obtaining a fusion tumor by a fusion tumor production method using a normal non-human animal, and culturing it, whereby the accumulated antibody is produced in the culture supernatant .

The CH as the recombinant antibody may be any one belonging to human immunoglobulin, preferably human immunoglobulin G (hIgG) type. Furthermore, any subtypes such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class can be used. In addition, as the CL of the recombinant antibody, any one belonging to human immunoglobulin may be used, and κ type or λ type can be used.

In the present invention, the so-called bispecific antibody refers to a polypeptide or protein that specifically binds to two different epitopes, respectively. Each antigen-binding site of a bispecific antibody can bind to different epitopes of a single antigen or to different antigens.

In the present invention, the so-called antigen-binding domain refers to a partial structure of a bispecific antibody having the function of specifically recognizing and binding to an antigen. Examples of the antigen-binding domain of the present invention include antibodies or fragments thereof, recombinant proteins comprising CDRs of antibodies, variable regions of antibodies comprising CDRs, ligands, receptors, and other proteins having the ability to bind to antigens. Recombinant protein or polypeptide. Among them, in the present invention, the antigen-binding domain is preferably the Fab of an antibody. In addition, the antigen-binding domain of the present invention also includes the VH of an individual antibody having antigen-binding activity.

In the present invention, the so-called first antigen-binding domain refers to the first antigen-binding domain contained in the bispecific antibody, and the so-called second antigen-binding domain refers to the first antigen-binding domain contained in the bispecific antibody. Antigen binding domains of different epitopes.

In particular, in the case of the bispecific antibody shown in (A) to (C) of FIG. 1 , which is a form of the bispecific antibody of the present invention, the first antigen-binding domain refers to the antigen-binding domain on the N-terminal side. , the second antigen-binding domain refers to the antigen-binding domain on the C-terminal side of the first antigen-binding domain.

In the present invention, the binding of the polypeptide, the antibody or the antibody fragment or the bispecific antibody or the bispecific antibody fragment to at least one of GPC3 and TfR can be achieved, for example, by using known immunological detection methods, preferably fluorescent It can be confirmed by a method for confirming the binding of cells expressing GPC3 or TfR to the antibody, such as by photocytometry. In addition, known immunological assays [Monoclonal Antibodies-Principles and Practice, 3rd edition, Academic Press (1996), Antibodies: Laboratory Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Laboratory Manual, Kodansha Scientific (1987)], etc. are used in combination.

In the present invention, the antigen binding domain that binds to GPC3 or TfR can be any one that specifically recognizes and binds to GPC3 or TfR. For example, it can be antibodies, ligands, receptors and naturally occurring interacting molecules, polypeptides, protein molecules and fragments thereof that can be produced by genetic recombination technology, as well as low molecules of the protein molecules or conjugates with natural substances, etc. any form.

As the bispecific antibody or the bispecific antibody fragment of the present invention, for example, a bispecific antibody or the bispecific antibody fragment having TfR internalization and/or decomposition activity can be exemplified. The bispecific antibody or the bispecific antibody fragment of the present invention preferably does not exhibit TfR internalization and/or decomposition activity in cells that do not express GPC3, and exhibits TfR internalization and/or only in cells expressing GPC3. Or break down the active bispecific antibody or the bispecific antibody fragment. The bispecific antibody or the bispecific antibody fragment selectively exhibits TfR internalization and/or catabolism activity on causative cells such as cancer cells expressing GPC3, and thus does not generate non-specific TfR internalization and/or It is preferable in this point that the side effects accompanying the decomposition are eliminated.

The bispecific antibody or the bispecific antibody fragment of the present invention can bind to GPC3 and TfR expressed on the same cell, or can bind to GPC3 and TfR expressed on different cells, preferably GPC3 and TfR expressed on the same cell TfR binders.

The bispecific antibody or the bispecific antibody fragment of the present invention is preferably one that inhibits the proliferation and/or induces cell death of target cells expressing GPC3 by inducing the internalization and/or decomposition of TfR.

The bispecific antibody or the bispecific antibody fragment of the present invention preferably does not inhibit the proliferation of cells that do not express GPC3 and express TfR and/or does not induce cell death, inhibits the proliferation of cells that co-express GPC3 and TfR, and / or induce cell death.

As mentioned above, it has been reported that TfR is expressed in cells including normal cells, on the other hand, GPC3 is expressed in hepatocellular carcinoma, malignant melanoma, ovarian clear cell carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, liver It is manifested in cancer cells such as blastoma, Wilms tumor, testicular germ cell tumor and liposarcoma. Thus, a bispecific antibody or bispecific antibody fragment that does not inhibit proliferation and/or induce cell death of cells that do not express GPC3 and TfR, inhibit proliferation and/or induce cell death of cells that co-express GPC3 and TfR It is suitable for use as a therapeutic drug that specifically inhibits only cancer cells without inhibiting the proliferation of normal cells.

The internalization and/or decomposition activity of TfR possessed by the bispecific antibody of the present invention or the bispecific antibody fragment can be confirmed by evaluating the amount of TfR protein on the cell surface and in the cells expressing TfR.

That is, as the bispecific antibody or the bispecific antibody fragment of the present invention, specifically, the bispecific antibody or the bispecific antibody fragment that induces the decomposition of TfR when it binds to both GPC3 and TfR can be exemplified. Wait.

The number of binding domains for a certain antigen contained in a molecule of bispecific antibody is called the valence of binding. For example, in the present invention, when a molecule of bispecific antibody contains two antigen-binding domains that bind to GPC3 and two antigen-binding domains that bind to TfR, the bispecific antibody is bivalent to GPC3 and GPC3, respectively. TfR binding.

In addition, the bispecific antibody of the present invention also includes a bispecific antibody comprising a plurality of antigen-binding domains bound via a suitable linker such as a linker comprising an immunoglobulin region or a fragment thereof.

The bispecific antibody of the present invention can be produced by existing techniques ([Nature Protocols, 9, 2450-2463 (2014)], International Publication No. 1998/050431, International Publication No. 2001 /7734, International Publication No. 2002/002773 and International Publication No. 2009/131239), etc.

In the present invention, the so-called immunoglobulin region is a minimum unit of a peptide containing about 100 amino acid residues having an amino acid sequence similar to that of an immunoglobulin and having at least 2 cysteine residues. In the present invention, the immunoglobulin domain also includes a polypeptide comprising a plurality of the above-mentioned minimum units of the immunoglobulin domain. Examples of the immunoglobulin region include VH, CH1, CH2 and CH3 of the heavy chain of immunoglobulins, and VL and CL of the light chain of immunoglobulins.

The animal species of the immunoglobulin is not particularly limited, but human is preferred. In addition, the isotype of the constant region of the immunoglobulin heavy chain can be any of IgD, IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgE, and IgG source and IgM source can be preferably exemplified. In addition, the isotype of the constant region of the immunoglobulin light chain may be any of κ and λ.

In addition, immunoglobulin domains also exist in proteins other than immunoglobulins. For example, major histocompatibility antigens (MHC), CD1, B7, and T cell receptors (TCRs) belong to the immunoglobulin superfamily. The immunoglobulin region contained in the protein. As the immunoglobulin region used in the bispecific antibody of the present invention, various immunoglobulin regions can be used.

In the case of human IgG, CH1 refers to the region comprising the amino acid sequence from positions 118 to 215 shown in the EU index. Likewise, CH2 refers to the region comprising the amino acid sequence from positions 231 to 340 shown in the EU index of Kabat et al., and CH3 refers to the region comprising positions 341 to 447 shown in the EU index of Kabat et al. The region of the amino acid sequence at the position. A flexible amino acid region called a hinge region (hereinafter sometimes also referred to as a hinge) exists between CH1 and CH2. The hinge region refers to the region comprising the amino acid sequence from positions 216 to 230 shown in the EU index of Kabat et al.

With regard to CL, in the case of the kappa chain of a human antibody, it refers to the region comprising the amino acid sequence from positions 108 to 214 shown in the Kabat numbering system, and in the case of the λ chain, it refers to the region comprising positions 108 to 214 The region of the amino acid sequence at position 215.

In the present invention, the so-called IgG moiety refers to a partial structure comprising IgG or an IgG modified with an Fc moiety constituting the bispecific antibody of the present invention, and the IgG moiety has an allotype two comprising one light chain and one heavy chain. A heterotetrameric structure formed by the association of two polymers.

The heavy chain constant region of the above-mentioned IgG part can be any subtype of IgG1, IgG2, IgG3, IgG4. Also, deletions, additions, substitutions and/or insertions can be performed locally to these amino acid sequences. In addition, the amino acid sequence of the heavy chain of IgG including CH1, hinge, CH2 and CH3 can be used in its entirety, or a partial fragment can be used in an appropriate combination. In addition, these amino acid sequences which are partially deleted, or these sequences may be used in an exchanged order. The subtype of IgG used in the IgG part is not particularly limited, but it is preferable that the Ser residue at position 228 of the heavy chain constant region of IgG4 is substituted with Pro, and the Leu residue at position 235 is substituted with Asn. The IgG4 variant (hereafter referred to as IgG4PE), or the heavy chain constant region of IgG4 where the Ser residue at position 228 is replaced by Pro, the Leu residue at position 235 is replaced by Asn, and the Arg residue at position 409 The IgG4 variant in which the base was substituted with Lys (hereinafter referred to as IgG4PE R409K).

For example, the heavy chain constant region of the above-mentioned IgG part (CH1-hinge-CH2-CH3 in this order from the N-terminal side) is preferably the heavy chain constant region of IgG4PE R409K comprising the amino acid sequence represented by SEQ ID NO: 255.

The two variable domains contained in the IgG portion of the present invention preferably recognize the same antigen. Moreover, it is preferable to have the same structure and amino acid sequence.

The bispecific antibody or antibody fragment thereof of the present invention also includes deletion, addition, substitution or insertion of one or more amino acid residues in the amino acid sequence constituting the bispecific antibody or the antibody fragment of the present invention, and An antibody or antibody fragment thereof having the same activity as the above-mentioned antibody or antibody fragment thereof.

The number of deletions, substitutions, insertions and/or additions of amino acids is 1 or more as determined by Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989), Laboratory Manual of Molecular Biology, John Wiley International (1987-1997), Nucleic Acids Research, 10, 6487(1982), Proceedings of the National Academy of Sciences, 79, 6409(1982), Genes , 34, 315 (1985), "Nucleic Acid Research", 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc. The deletion that can be realized by well-known techniques such as site-directed mutagenesis The number of degrees of , substitution, insertion or addition is not particularly limited. For example, it is usually 1 to several tens of pieces, preferably 1 to 20 pieces, more preferably 1 to 10 pieces, and still more preferably 1 to 5 pieces.

The above-mentioned so-called deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence of the bispecific antibody of the present invention means that there are one or more amino acid residues at any position of the amino acid sequence Deletion, substitution, insertion or addition of amino acid residues. In addition, there are cases where deletion, substitution, insertion or addition occur at the same time, and the amino acid residues for substitution, insertion or addition are either natural or non-natural.

As natural amino acid residues, for example, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L- -Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Arginine, L-Methionine, L-Phenylalanine, L-Proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and L-cysteine, etc.

Preferred examples of mutually replaceable amino acid residues are shown below. Amino acid residues included in the same group can be substituted for each other.

Group A: leucine, isoleucine, glycine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, O-methylserine, Tributylglycine, tert-butylalanine, cyclohexylalanine Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberate Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid Group E: Proline, 3-Hydroxyproline, 4-Hydroxyproline Group F: Serine, Threonine, Homoserine Group G: Phenylalanine, Tyrosine

The bispecific antibody or the antibody fragment of the present invention also includes antibodies comprising any amino acid residues that are post-translationally modified. As post-translational modifications, for example, deletion of a lysine residue at the C-terminus of the H chain [lysine clipping] and substitution of a glutamine residue at the N-terminus of a polypeptide with a pyro Glutamic acid (pyroGlu) et al [Beck et al, Analytical Chemistry, 85, 715-736 (2013)].

As the bispecific antibody of the present invention, for example, the bispecific antibody of the following (I) or (II) can be mentioned. (I) The C-terminus of the heavy chain of the IgG portion comprising the first antigen-binding domain and the C-terminus of the heavy chain comprising (a1) an IgG portion comprising a first antigen-binding domain and (b1) a second antigen-binding domain and the second antigen-binding domain A bispecific antibody in which one of the first antigen-binding domain and the second antigen-binding domain binds to GPC3 and the other binds to TfR (also referred to as a C-terminal bispecific antibody) is N-terminally linked. In the C-terminal bispecific antibody, the C-terminus of the heavy chain comprising the IgG portion of the first antigen-binding domain and the N-terminus of the second antigen-binding domain are preferably linked directly or via a linker, more preferably directly linked . (II) comprising (a2) an IgG portion comprising a second antigen-binding domain and (b2) a first antigen-binding domain, the C-terminus of the first antigen-binding domain and the heavy chain of the IgG portion comprising the second antigen-binding domain A bispecific antibody in which one of the first antigen-binding domain and the second antigen-binding domain binds to GPC3 and the other binds to TfR (also referred to as N-terminal bispecific antibody) is N-terminally linked. In the N-terminal type bispecific antibody, the C-terminus of the first antigen-binding domain and the N-terminus of the heavy chain comprising the IgG portion of the second antigen-binding domain are preferably linked directly or via a linker, more preferably directly linked.

In the present invention, the so-called linker may be any one, preferably a peptide chain. The amino acid sequence of the peptide chain includes, for example, a repeat sequence of ES, ESKYG, ESKYGPP, GGGGS, or GGGGS, or a partial or whole sequence of a constant region such as a hinge region and a CH1 domain of an antibody, etc. .

The bispecific antibody of the present invention may adopt any structure of the C-terminal bispecific antibody of the above (I) and the N-terminal bispecific antibody of the above (II).

As one form of the bispecific antibody of the above (I), a bispecific antibody in which the second antigen-binding domain is an Fab of an antibody can be exemplified. Also, a bispecific antibody in which the C-terminus of the heavy chain of the IgG moiety comprising the first antigen-binding domain is linked directly or via a linker to the N-terminus of the heavy chain of the Fab of the antibody serving as the second antigen-binding domain can also be exemplified. .

As one form of the bispecific antibody of the above (II), a bispecific antibody in which the first antigen-binding domain is the Fab of the antibody can be exemplified. Also, a bispecific antibody in which the C-terminus of the heavy chain of the Fab of the antibody serving as the first antigen-binding domain is linked directly or via a linker to the N-terminus of the heavy chain of the IgG portion of the second antigen-binding domain is linked. .

As one form of the bispecific antibody of the above (I) or (II), it is preferable that any one of the first antigen-binding domain and the second antigen-binding domain is an anti-GPC3 IgG antibody Fab (anti-GPC3 Fab).

As one form of the bispecific antibody of the above (I) or (II), it is preferable that either the first antigen-binding domain or the second antigen-binding domain is an anti-TfR IgG antibody Fab (anti-TfR Fab).

As one form of the bispecific antibody of the above (I) or (II), it is preferable that the first antigen-binding domain is anti-GPC3 Fab, and the second antigen-binding domain is anti-TfR Fab.

As one form of the bispecific antibody of the above (I) or (II), it is preferable that the first antigen-binding domain is anti-TfR Fab, and the second antigen-binding domain is anti-GPC3 Fab.

Specific examples of the bispecific antibody of the present invention include any bispecific antibody selected from the group consisting of the following (1) to (4). (1) A bispecific antibody comprising an IgG moiety comprising an anti-GPC3 Fab and an anti-TfR Fab, the C-terminus of the heavy chain comprising the IgG moiety of the anti-GPC3 Fab and the N-terminus of the heavy chain of the anti-TfR Fab being directly linked (C-terminal type GPC3-TfR bispecific antibody); (2) A bispecific antibody comprising an IgG portion comprising anti-TfR Fab and an anti-GPC3 Fab, the C-terminus of the heavy chain comprising the IgG portion of the anti-TfR Fab and the N-terminus of the heavy chain of the anti-GPC3 Fab being directly linked (C-terminal type TfR-GPC3 bispecific antibody); (3) A bispecific antibody comprising an IgG moiety comprising anti-GPC3 Fab and an anti-TfR Fab, the C-terminus of the heavy chain of the anti-TfR Fab and the N-terminus of the heavy chain comprising the IgG moiety of the anti-GPC3 Fab being directly linked (N-terminal type TfR-GPC3 bispecific antibody; (4) A bispecific antibody comprising an IgG moiety comprising an anti-TfR Fab and an anti-GPC3 Fab, the C-terminus of the heavy chain of the anti-GPC3 Fab and the N-terminus of the heavy chain comprising the IgG moiety of the anti-TfR Fab being directly linked (N-terminal type GPC3-TfR bispecific antibody).

The VL of the anti-TfR Fab and the VL of the anti-GPC3 Fab in the bispecific antibodies described in (1) to (4) above may be the same or different, but from the viewpoint of improving the stability of the structure and the stability of the reactivity, Preferably they are the same.

As an example of the anti-GPC3 Fab in the present invention, there can be exemplified a Fab having VH and VL, wherein the VH contains any one of CDRs 1 to 3 selected from the following (g1) to (g40), and the VL contains SEQ ID NO: 24, respectively. CDR1-3 of the amino acid sequence represented by ~26. (g1) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 91 to 93, (g2) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 95 to 97, (g3) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 99 to 101, (g4) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 103 to 105, (g5) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 107 to 109, (g6) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 111 to 113, (g7) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 115 to 117, (g8) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 119 to 121, respectively, (g9) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 123 to 125, (g10) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 127 to 129, (g11) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 131 to 133, (g12) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 135 to 137, (g13) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 139 to 141, (g14) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 143 to 145, (g15) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 147 to 149, (g16) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 151 to 153, (g17) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 155 to 157, (g18) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 159 to 161, (g19) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 163 to 165, (g20) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 167 to 169, (g21) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 171 to 173, (g22) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 175 to 177, (g23) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 179 to 181, (g24) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 183 to 185, (g25) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 187 to 189, (g26) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 191 to 193, (g27) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 195 to 197, (g28) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 199 to 201, (g29) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 203 to 205, (g30) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 207 to 209, (g31) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 211 to 213, (g32) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 215 to 217, (g33) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 219 to 221, (g34) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 223 to 225, (g35) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 227 to 229, (g36) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 231 to 233, (g37) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 235 to 237, respectively, (g38) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 239 to 241, respectively, (g39) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 243 to 245, respectively, (g40) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 247 to 249, respectively.

The anti-GPC3 Fab of the present invention also includes amino acid sequences comprising CDR1-3 of VH selected from any one of the above (g1)-(g40) and CDR1-3 of VL represented by SEQ ID NOs: 24-26, respectively at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical amino acid sequences of CDRs 1-3 of VH and VL to GPC3-binding Fab.

As an example of the anti-GPC3 Fab of the present invention, there may be exemplified: Any of 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246 The VH of the amino acid sequence shown in SEQ ID NO: 23, and the Fab containing the VL of the amino acid sequence shown in SEQ ID NO: 23.

An example of the anti-TfR Fab of the present invention includes a Fab having a VH and a VL, the VH containing any one of CDRs 1 to 3 selected from the following (t1) to (t16), and the VL contains SEQ ID NOs: 24 to 24, respectively. CDR1-3 of the amino acid sequence represented by 26. (t1) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 28 to 30, respectively, (t2) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 32 to 34, respectively, (t3) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 37 to 39, (t4) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 41 to 43, (t5) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 45 to 47, respectively, (t6) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 49 to 51, respectively, (t7) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 53 to 55, respectively, (t8) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 57 to 59, (t9) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 61 to 63, (t10) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 65 to 67, (t11) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 69 to 71, respectively, (t12) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 73 to 75, respectively, (t13) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 269 to 271, (t14) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 79 to 81, respectively, (t15) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 83 to 85, respectively, (t16) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 87 to 89, respectively.

The anti-TfR Fab of the present invention also includes amino acid sequences comprising CDR1-3 of VH selected from any one of the above (t1)-(t16) and CDR1-3 of VL represented by SEQ ID NOs: 24-26, respectively at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least Fab that binds to TfR of 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical amino acid sequences of CDRs 1-3 of VH and VL.

As an example of the anti-TfR Fab of the present invention, there may be exemplified: The VH of the amino acid sequence represented by any one of 86, and the Fab comprising the VL of the amino acid sequence represented by SEQ ID NO: 23.

The IgG part of the present invention contains a heavy chain constant region, and the subtype of the heavy chain constant region is not particularly limited, preferably human IgG, more preferably human IgG4 or a modified version thereof, and more preferably human IgG4 PE or Human IgG4 PE R409K. As a specific example of the amino acid sequence of the above-mentioned heavy chain constant region, the amino acid sequence represented by SEQ ID NO: 255 can be exemplified.

(A) of FIG. 1 is a diagram showing the structures of an N-terminal GPC3-TfR bispecific antibody and an N-terminal TfR-GPC3 bispecific antibody. As shown in (A) of FIG. 1 , the N-terminal GPC3-TfR bispecific antibody and the N-terminal TfR-GPC3 bispecific antibody contain two VH1 (from the N-terminal side) in order from the N-terminal side. VH at the 1st position), CH1, VH2 (VH at the 2nd position from the N-terminal side), CH1' (CH1 at the 2nd position from the N-terminal side), hinge, polypeptide chains of CH2 and CH3, and 4 A bispecific antibody comprising polypeptide chains of VL and CL in order from the N-terminal side. In the N-terminal GPC3-TfR bispecific antibody, VH1 is the VH of the anti-GPC3 antibody, and VH2 is the VH of the anti-TfR antibody. In addition, in the N-terminal TfR-GPC3 bispecific antibody, VH1 is the VH of the anti-TfR antibody, and VH2 is the VH of the anti-GPC3 antibody.

(B) of FIG. 1 is a diagram showing the structures of the C-terminal GPC3-TfR bispecific antibody and the C-terminal TfR-GPC3 bispecific antibody. As shown in FIG. 1(B), the C-terminal GPC3-TfR bispecific antibody and the C-terminal TfR-GPC3 bispecific antibody are composed of two strips including VH1, CH1, hinge, and CH2 in this order from the N-terminal side. , CH3, VH2 and CH1' polypeptide chains, and 4 bispecific antibodies comprising VL and CL polypeptide chains in sequence from the N-terminal side. In the C-terminal GPC3-TfR bispecific antibody, VH1 is the VH of the anti-GPC3 antibody, and VH2 is the VH of the anti-TfR antibody. Furthermore, in the C-terminal TfR-GPC3 bispecific antibody, VH1 is the VH of the anti-TfR antibody, and VH2 is the VH of the anti-GPC3 antibody.

In the present invention, the N-terminal GPC3-TfR bispecific antibody and the N-terminal TfR-GPC3 bispecific antibody include VH1 (the first VH from the N-terminal side), CH1, VH2 (VH at the 2nd position from the N-terminal side), CH1' (CH1 at the 2nd position from the N-terminal side), hinge, polypeptide chains of CH2 and CH3, and C-terminal type GPC3-TfR bispecific The polypeptide chain of the antibody and the C-terminal TfR-GPC3 bispecific antibody comprising VH1, CH1, hinge, CH2, CH3, VH2 and CH1' in order from the N-terminal side is called the heavy chain of the bispecific antibody.

In addition, the N-terminal GPC3-TfR bispecific antibody, the N-terminal TfR-GPC3 bispecific antibody, the C-terminal GPC3-TfR bispecific antibody, and the C-terminal TfR-GPC3 bispecific antibody were obtained from the N-terminal Polypeptide chains flanking VL and CL in sequence are called the light chains of bispecific antibodies.

The two heavy chains of bispecific antibodies are associated by the formation of disulfide bonds between cysteine residues in the hinge region. The heavy and light chains of bispecific antibodies are associated by disulfide bond formation between the cysteine residues of CH1 or CH1' and CL. The two heavy chains of the bispecific antibody may be the same or different, preferably the same. In addition, the four light chains of the bispecific antibody may be different from each other or the same, and preferably all of the four light chains are the same. In addition, CL may be any of a Cλ chain and a Cκ chain.

Anti-GPC3 antibody among the above-mentioned N-terminal GPC3-TfR bispecific antibody, N-terminal TfR-GPC3 bispecific antibody, C-terminal GPC3-TfR bispecific antibody, and C-terminal TfR-GPC3 bispecific antibody The VH can be exemplified: including SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, of amino acid sequences represented by any one of 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246 VH. From the viewpoint of enhancing the cell proliferation inhibitory activity of the bispecific antibody, among these, it is preferable to include a compound selected from the group consisting of SEQ ID NOs: 94, 98, 102, 106, 114, 130, 166, 178, 186 and 190 The VH of the amino acid sequence represented by any one is more preferably the VH comprising the amino acid sequence represented by any one of SEQ ID NOs: 94, 114 and 190, and more preferably the amine represented by SEQ ID NO: 190 The VH of the amino acid sequence.

Also, as an antibody in the above-mentioned N-terminal GPC3-TfR bispecific antibody, N-terminal TfR-GPC3 bispecific antibody, C-terminal GPC3-TfR bispecific antibody, and C-terminal TfR-GPC3 bispecific antibody The VH of the TfR antibody includes, for example, any one of SEQ ID NOs: 27, 35, 31, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 268, 78, 82, and 86. The VH of the amino acid sequence. From the viewpoint of enhancing the cell growth inhibitory activity of the bispecific antibody, among these, a VH comprising the amino acid sequence represented by SEQ ID NO: 35 or 40 is preferred.

The VL of the N-terminal GPC3-TfR bispecific antibody, N-terminal TfR-GPC3 bispecific antibody, C-terminal GPC3-TfR bispecific antibody and C-terminal TfR-GPC3 bispecific antibody of the present invention may be the same Although different, from the viewpoint of improving the stability of the structure and the stability of the reactivity, the same is preferable. As VL, the VL containing the amino acid sequence shown in SEQ ID NO: 23 can be mentioned, for example. Moreover, as CL, the CL containing the amino acid sequence represented by SEQ ID NO: 272 can be mentioned, for example.

As an example of the N-terminal GPC3-TfR bispecific antibody of the present invention, the following can be exemplified: (N1) a bispecific antibody comprising 2 heavy chains and 4 light chains, wherein the VH1 of the heavy chain comprises SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134,138,142,146,150,154,158,162,166,178,186,190,194,198,202,206,210,214,218,222,226,230,234,238,242 and The VH of the amino acid sequence represented by any one of 246, VH2 includes SEQ ID NOs: 35, 31, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 268, 78, 82 and 86 The VH of the amino acid sequence represented by any one, and the VL of the above-mentioned light chain is the VL comprising the amino acid sequence represented by SEQ ID NO: 23. (N2) A bispecific antibody comprising 2 heavy chains and 4 light chains, the two heavy chains respectively contain SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, VH of the amino acid sequence represented by any one of 230, 234, 238, 242 and 246, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, comprising SEQ ID NOs: 35, 31, 36, 40, 44, 48, The VH of the amino acid sequence represented by any one of 52, 56, 60, 64, 68, 72, 268, 78, 82 and 86, and the CH comprising the amino acid sequence represented by SEQ ID NO: 255, the four amino acid sequences The chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272, respectively, from the N-terminal side.

Among the above-mentioned N-terminal GPC3-TfR bispecific antibodies, the following ones are preferred from the viewpoint of cell growth inhibitory activity. (N1-1) A bispecific antibody comprising 2 heavy chains and 4 light chains, the VH1 of the heavy chain is SEQ ID NO: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, The VH of the amino acid sequence represented by any one of 242 and 246, VH2 is the VH comprising the amino acid sequence represented by SEQ ID NO: 35, and the VL of the above-mentioned light chain is the VL comprising the amino acid sequence represented by SEQ ID NO: 23. Among these, more preferred are bispecific antibodies comprising two heavy chains and four light chains, wherein the VH1 of the heavy chain is SEQ ID NOs: 94, 98, 102, 114, 130, 166, 178, The VH of the amino acid sequence represented by any one of 186 and 190, VH2 is the VH comprising the amino acid sequence represented by SEQ ID NO: 35, and the VL of the above-mentioned light chain is the VL comprising the amino acid sequence represented by SEQ ID NO: 23. More preferably, it is a bispecific antibody comprising 2 heavy chains and 4 light chains, wherein the VH1 of the heavy chain is a VH, VH2 comprising the amino acid sequence represented by any one of SEQ ID NOs: 94, 114 and 190. The VL of the above-mentioned light chain is the VL comprising the amino acid sequence represented by SEQ ID NO: 23, which is the VH comprising the amino acid sequence represented by SEQ ID NO: 35. Particularly preferred is a bispecific antibody comprising two heavy chains and four light chains, wherein VH1 of the heavy chain is a VH comprising the amino acid sequence represented by SEQ ID NO: 190, and VH2 is a VH2 comprising an amino acid sequence represented by SEQ ID NO: 35 The VH of the acid sequence and the VL of the above-mentioned light chain are the VL comprising the amino acid sequence represented by SEQ ID NO: 23. (N1-2) A bispecific antibody comprising 2 heavy chains and 4 light chains, the VH1 of the heavy chain is SEQ ID NO: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, The VH of the amino acid sequence represented by any one of 242 and 246, VH2 is the VH comprising the amino acid sequence represented by SEQ ID NO: 40, and the VL of the above-mentioned light chain is the VL comprising the amino acid sequence represented by SEQ ID NO: 23. Among these, more preferred is a bispecific antibody comprising two heavy chains and four light chains, wherein the VH1 of the heavy chain comprises SEQ ID NOs: 94, 98, 102, 106, 114, 130, 178 and The VH of the amino acid sequence represented by any one of 190, VH2 is the VH comprising the amino acid sequence represented by SEQ ID NO: 40, and the VL of the above-mentioned light chain is the VL comprising the amino acid sequence represented by SEQ ID NO: 23. More preferably, it is a bispecific antibody comprising 2 heavy chains and 4 light chains, wherein the VH1 of the heavy chain is a VH, VH2 comprising the amino acid sequence represented by any one of SEQ ID NOs: 94, 114 and 190. The VL of the above-mentioned light chain is the VL comprising the amino acid sequence represented by SEQ ID NO: 23, which is the VH comprising the amino acid sequence represented by SEQ ID NO: 40. Particularly preferred is a bispecific antibody comprising two heavy chains and four light chains, wherein VH1 of the heavy chain is a VH comprising the amino acid sequence represented by SEQ ID NO: 190, and VH2 is a VH2 comprising an amino acid sequence represented by SEQ ID NO: 40 The VH of the acid sequence and the VL of the above-mentioned light chain are the VL comprising the amino acid sequence represented by SEQ ID NO: 23.

(N2-1) A bispecific antibody comprising 2 heavy chains and 4 light chains, the two heavy chains respectively contain SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, VH of the amino acid sequence represented by any one of 226, 230, 234, 238, 242 and 246, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and CH comprising the amino acid sequence represented by SEQ ID NO: 255, the four light chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and the amino acid represented by SEQ ID NO: 272 from the N-terminal side, respectively CL of the sequence. (N2-2) A bispecific antibody comprising 2 heavy chains and 4 light chains, the two heavy chains respectively contain SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, VH of the amino acid sequence represented by any one of 226, 230, 234, 238, 242 and 246, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 40, and CH comprising the amino acid sequence represented by SEQ ID NO: 255, the four light chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and the amino acid represented by SEQ ID NO: 272 from the N-terminal side, respectively CL of the sequence.

From the viewpoint of cell growth inhibitory activity, among the N-terminal GPC3-TfR bispecific antibodies of (N1-1), (N1-2), (N2-1) and (N2-2), the preferred The N-terminal GPC3-TfR bispecific antibody for VH1 is the VH comprising the amino acid sequence represented by any one of SEQ ID NOs: 94, 98, 102, 106, 114, 130, 166, 178, 186 and 190. The N-terminal GPC3-TfR bispecific antibody showed excellent cell proliferation activity even in cells with moderate to low levels of GPC3 expression. As described above, there are differences in the expression level of GPC3 in cancer cells of different cancer patients, and the expression is not limited to high expression, and some cancer patients have low to moderate expression. Therefore, it is considered that the above-mentioned N-terminal GPC3-TfR bispecific antibody, which exhibits excellent cell proliferation activity even in cells expressing GPC3 at a moderate to low level, also has excellent properties from a clinical point of view.

Among the above-mentioned N-terminal GPC3-TfR bispecific antibodies, from the viewpoint of the stability of physical properties and growth inhibitory activity, the following are more preferable: (N1-2-1) a bispecific antibody comprising 2 heavy chains and 4 light chains, wherein the VH1 of the heavy chain is a VH comprising the amino acid sequence represented by any one of SEQ ID NOs: 94, 114 and 190, VH2 is a VH comprising the amino acid sequence represented by SEQ ID NO: 40, and the VL of the above-mentioned light chain is a VL comprising the amino acid sequence represented by SEQ ID NO: 23. (N2-2-1) A bispecific antibody comprising 2 heavy chains and 4 light chains, the two heavy chains respectively contain any one of SEQ ID NOs: 94, 114 and 190 in sequence from the N-terminal side. VH of the amino acid sequence represented by SEQ ID NO: 253, CH1 comprising the amino acid sequence represented by SEQ ID NO: 40, VH comprising the amino acid sequence represented by SEQ ID NO: 40, and CH comprising the amino acid sequence represented by SEQ ID NO: 255, the 4 The light chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272, respectively, from the N-terminal side.

Among the above-mentioned N-terminal GPC3-TfR bispecific antibodies, from the viewpoint of cell growth inhibitory activity, the following are particularly preferred: (N1-2-2) A bispecific antibody comprising 2 heavy chains and 4 light chains, the VH1 of the heavy chain is the VH comprising the amino acid sequence represented by SEQ ID NO: 190, and the VH2 is comprising the amino acid sequence represented by SEQ ID NO: 40 The VH of the amino acid sequence and the VL of the above-mentioned light chain are the VL comprising the amino acid sequence represented by SEQ ID NO: 23. (N2-2-2) A bispecific antibody comprising 2 heavy chains and 4 light chains, the two heavy chains respectively contain a VH comprising the amino acid sequence represented by SEQ ID NO: 190 from the N-terminal side, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 40, and CH comprising the amino acid sequence represented by SEQ ID NO: 255, the four light chains are respectively from the N-terminal side It contains VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 in this order.

Specific examples of the N-terminal GPC3-TfR bispecific antibody of the present invention include Nt-G3042-TfR1071, Nt-G1048-TfR1071, Nt-G1104-TfR1071, Nt- G4045-TfR1071, Nt-G4068-TfR1071, Nt-G2072-TfR1071, Nt-G1018-TfR1071, Nt-G2038-TfR1071, Nt-G1054-TfR1071, Nt-G1016-TfR1071, Nt-G3071, Nt-G119-T TfR1071, Nt-G2103-TfR1071, Nt-G2067-TfR1071, Nt-G2133-TfR1071, Nt-G1036-TfR1071, Nt-G2002-TfR1071, Nt-G1015-TfR1071, Nt-G4033-TfR1071, Nt-G202025-TfR1071 Nt-G2011-TfR1071, Nt-G1122-TfR1071, Nt-G4037-TfR1071, Nt-G3033-TfR1071, Nt-G3008-TfR1071, Nt-G4009-TfR1071, Nt-G4025-TfR1071, Nt-fR1071, Nt-G1107 G2017NNA-TfR1071、Nt-G1137-TfR1071、Nt-G2024NNA-TfR1071、Nt-G3177NTA-TfR1071、Nt-G3062NYA-TfR1071、Nt-G3018-TfR1071、Nt-G3024-TfR1071、Nt-G3117-TfR1071、Nt-G4025- TfR1071_202、Nt-G2038-TfR1071_202、Nt-G1036-TfR1071_202、Nt-G2002-TfR1071_202、Nt-G1119-TfR1071_202、Nt-G1137-TfR1071_202、Nt-G2133-TfR1071_202、Nt-G2067-TfR1071_202及圖9A～圖9G中Bispecific antibodies described.

From the viewpoint of stability of physical properties and cell growth inhibitory activity, among the above-mentioned N-terminal GPC3-TfR bispecific antibodies, Nt-G4025-TfR1071, Nt-G2038-TfR1071, Nt-G1036-TfR1071, , NT-G1119-TFR1071, NT-G1137-TFR1071, NT-G2133-TFR1071, NT-G1107-TFR1071, NT-G307-TFR1071, NT-G407-TFR1071, nt G1036-TfR1071_202、Nt-G2002-TfR1071_202、Nt-G1119-TfR1071_202、Nt-G1137-TfR1071_202、Nt-G2133-TfR1071_202及Nt-G2067-TfR1071_202，更佳為Nt-G4025-TfR1071、Nt-G2002-TfR1071、Nt -G1119-TfR1071、Nt-G4025-TfR1071_202、Nt-G2002-TfR1071_202及Nt-G1119-TfR1071_202，尤佳為Nt-G4025-TfR1071_202、Nt-G2002-TfR1071_202及Nt-G1119-TfR1071_202，最佳為Nt-G4025 -TfR1071_202.

These N-terminal GPC3-TfR bispecific antibodies are named according to the manner of Nt-[the clone name of the anti-GPC3 antibody]-[the clone name of the anti-TfR antibody], indicating that they contain 2 heavy chains and 4 light chains The bispecific antibody, the two heavy chains sequentially contain the VH of the anti-GPC3 antibody, the CH1 containing the amino acid sequence represented by SEQ ID NO: 253, the VH of the anti-TfR antibody, and the VH of the anti-TfR antibody from the N-terminal side. CH of the amino acid sequence represented by 255, the four light chains contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 in order from the N-terminal side.

The sequence number of the amino acid sequence of the VH of the anti-GPC3 antibody is shown in Tables 4 and 5. In addition, the SEQ ID NOs of the amino acid sequence of the VH of the anti-TfR antibody are shown in Tables 1 to 3. Here, as the VH of TfR1071, the VH containing the amino acid sequence shown in SEQ ID NO: 35 was used. As the VL of the anti-TfR antibody and the GPC3 antibody, the VL containing the amino acid sequence shown in SEQ ID NO: 23 was used. For example, Nt-G3042-TfR1071 represents a bispecific antibody comprising two heavy chains and four light chains, and the two heavy chains contain, in order from the N-terminal side, a VH comprising the amino acid sequence represented by SEQ ID NO: 206, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and CH comprising the amino acid sequence represented by SEQ ID NO: 255, the four light chains from the N-terminal side VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 are contained in this order.

As an example of the C-terminal GPC3-TfR bispecific antibody of the present invention, the following can be exemplified: (C1) a bispecific antibody comprising 2 heavy chains and 4 light chains, wherein the VH1 of the heavy chain comprises SEQ ID NOs: 90, 142, 126, 178, 198, 146, 218, 150, 202, 226, 94, The amino acid sequence represented by any one of 98, 102, 106, 110, 114, 118, 122, 130, 134, 138, 154, 158, 162, 234, 238, 242, 246, 186, 190 and 194 VH and VH2 are VH comprising the amino acid sequence represented by SEQ ID NO: 35, and the VL of the above-mentioned light chain is VL comprising the amino acid sequence represented by SEQ ID NO: 23. (C2) a bispecific antibody comprising 2 heavy chains and 4 light chains, the two heavy chains respectively contain SEQ ID NOs: 90, 142, 126, 178, 198, 146, 218, Any of 150, 202, 226, 94, 98, 102, 106, 110, 114, 118, 122, 130, 134, 138, 154, 158, 162, 234, 238, 242, 246, 186, 190 and 194 VH of the amino acid sequence represented by SEQ ID NO: 255, CH comprising the amino acid sequence represented by SEQ ID NO: 35, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and CH1 of the amino acid sequence represented by SEQ ID NO: 253, the 4 The light chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272, respectively, from the N-terminal side.

Specific examples of the C-terminal side GPC3-TfR bispecific antibody of the present invention include Ct-G2025-TfR1071, Ct-G1048-TfR1071, Ct-G1104-TfR1071, Ct- G1122-TfR1071, Ct-G4045-TfR1071, Ct-G3008-TfR1071, Ct-G1137-TfR1071, Ct-G1054-TfR1071, Ct-G4068-TfR1071, Ct-G1015-TfR1071 and bispecific antibodies described in FIG. 8 .

These C-terminal GPC3-TfR bispecific antibodies are named according to the manner of Ct-[the clone name of the anti-GPC3 antibody]-[the clone name of the anti-TfR antibody], indicating that they contain 2 heavy chains and 4 light chains The bispecific antibody, the two heavy chains sequentially contain the VH of the anti-GPC3 antibody, the CH containing the amino acid sequence represented by SEQ ID NO: 255, the VH of the anti-TfR antibody, and the VH of the anti-TfR antibody from the N-terminal side. CH1 of the amino acid sequence represented by 253, the 4 light chains contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 in order from the N-terminal side.

The sequence number of the amino acid sequence of the VH of the anti-GPC3 antibody is shown in Tables 4 and 5. In addition, the SEQ ID NOs of the amino acid sequence of the VH of the anti-TfR antibody are shown in Tables 1 to 3. Here, as the VH of TfR1071, the VH containing the amino acid sequence shown in SEQ ID NO: 35 was used. As the VL of the anti-TfR antibody and the GPC3 antibody, the VL containing the amino acid sequence shown in SEQ ID NO: 23 was used. For example, Ct-G2025-TfR1071 represents a bispecific antibody comprising two heavy chains and four light chains, and the two heavy chains contain, in order from the N-terminal side, a VH comprising the amino acid sequence represented by SEQ ID NO: 226, The 4 light chains from the N-terminal side include CH comprising the amino acid sequence represented by SEQ ID NO: 255, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and CH1 comprising the amino acid sequence represented by SEQ ID NO: 253 VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 are contained in this order.

As one form of the N-terminal bispecific antibody of the present invention, for example, the following bispecific antibodies can be exemplified. ・Contains an IgG moiety comprising an anti-GPC3 Fab, and a VH of an anti-TfR antibody that binds to TfR alone, the C-terminus of the VH of the anti-TfR antibody is connected to an IgG moiety comprising an anti-GPC3 Fab via a linker (GGGGS described in SEQ ID NO: 256) A bispecific antibody linked to the N-terminus of the heavy chain (TfR-GS-GPC3 bispecific antibody); or ・Contains the IgG portion comprising anti-TfR Fab and the VH of the anti-GPC3 antibody alone and GPC3, the C-terminus of the VH of the anti-GPC3 antibody is combined with the IgG portion comprising the anti-TfR Fab via a linker (GGGGS described in SEQ ID NO: 256) A bispecific antibody linked to the N-terminus of the chain (GPC3-GS-TfR bispecific antibody).

(C) of FIG. 1 is a diagram showing the structures of the GPC3-GS-TfR bispecific antibody and the TfR-GS-GPC3 bispecific antibody. As shown in (C) of FIG. 1 , the GPC3-GS-TfR bispecific antibody and the TfR-GS-GPC3 bispecific antibody contain two VH1 and GS sequences [GGGGS (SEQ ID NO: 256) from the N-terminal side in this order. )], VH2, CH1, hinge, CH2 and CH3 polypeptide chains, 2 polypeptide chains including VL and CL sequentially from the N-terminal side. In the GPC3-GS-TfR bispecific antibody, VH1 is the VH of the anti-GPC3 antibody bound to GPC3 alone, and VH2 is the VH of the anti-TfR antibody. In the TfR-GS-GPC3 bispecific antibody, VH1 is the VH of the anti-TfR antibody bound to TfR alone, and VH2 is the VH of the anti-GPC3 antibody.

In the present invention, the heavy chain and light chain of the bispecific antibody respectively contain the VH1 and GS sequences from the N-terminal side of the GPC3-GS-TfR bispecific antibody and the TfR-GS-GPC3 bispecific antibody [ GGGGS (SEQ ID NO: 256)], a polypeptide chain of VH2, CH1, hinge, CH2 and CH3, and a polypeptide chain comprising VL and CL in order from the N-terminal side.

In the GPC3-GS-TfR bispecific antibody and the TfR-GS-GPC3 bispecific antibody, the two heavy chains are associated through disulfide bond formation by cysteine residues in the hinge region. In addition, the heavy chain and the light chain are associated by the formation of disulfide bonds between the cysteine residues of CH1 and CL.

As an example of the GPC3-GS-TfR bispecific antibody of the present invention, the following can be exemplified: (GS1) A bispecific antibody comprising two heavy chains and two light chains, the VH1 of the heavy chain is the VH comprising the amino acid sequence represented by SEQ ID NO: 250, and the VH2 is the amino acid sequence represented by SEQ ID NO: 27 The VH of the above-mentioned light chain is the VL comprising the amino acid sequence represented by SEQ ID NO: 23. (GS2) A bispecific antibody comprising two heavy chains and two light chains, the two heavy chains respectively contain a VH comprising the amino acid sequence represented by SEQ ID NO: 250 from the N-terminal side, and a VH comprising the amino acid sequence represented by SEQ ID NO: 256, respectively. The linker of the amino acid sequence shown in SEQ ID NO: 27, the VH including the amino acid sequence shown in SEQ ID NO: 27, and the CH including the amino acid sequence shown in SEQ ID NO: 255, the two light chains respectively contain sequentially from the N-terminal side VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272.

Specific examples of the GPC3-GS-TfR bispecific antibody of the present invention include HN3-GS-TfR1071 described in the following Examples.

The bispecific antibody or the antibody fragment of the present invention also includes an antibody or the antibody fragment having effector activity.

The so-called effector activity refers to the antibody-dependent cytotoxic activity mediated by the Fc region of the antibody, for example, antibody-dependent cytotoxic activity (Antibody-Dependent Cellular Cytotoxicity activity; ADCC activity), complement-dependent cytotoxic activity (Complement-dependent cytotoxic activity -Dependent Cytotoxicity activity; CDC activity), antibody-dependent phagocytic activity of phagocytes such as macrophages or dendritic cells (Antibody-dependent cellular phagocytosis activity; ADCP activity) and prophagocytosis.

In the present invention, ADCC activity and CDC activity can be measured by a known measurement method ["Cancer Immunol. Immunother.", 36, 373 (1993)].

The so-called ADCC activity refers to the activity of activating immune cells (natural killer cells, etc.) to kill the target cells after the antibody binds to the antigen on the target cell and then binds to the Fc receptor of the immune cell through the Fc region of the antibody.

An Fc receptor (FcR) is a receptor that binds to the Fc region of an antibody, and induces various effector activities through the binding of the antibody. Each FcR corresponds to an antibody subtype, and IgG, IgE, IgA, and IgM specifically bind to FcγR, FcεR, FcαR, and FcμR, respectively. Furthermore, FcγR exists in the subtypes of FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), and each subtype has the isoforms of FcγRIA, FcγRIB, FcγRIC, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA and FcγRIIIB. These different FcyRs are present on different cells [Annu. Rev. Immunol., 9:457-492 (1991)]. In humans, FcγRIIIB is specifically expressed in neutrophils, and FcγRIIIA is expressed in monocytes, natural killer cells (NK cells), macrophages and some T cells. NK cell-dependent ADCC activity is induced by antibody binding to FcγRIIIA.

The so-called CDC activity refers to the activity of killing target cells by activating a series of cascade reactions (complement activation pathway) including complement-related protein groups in the blood after the antibody binds to the antigen on the target cell. Also, protein fragments produced by activating complement induce migration and activation of immune cells. The cascade of CDC activity is as follows: first, C1q binds to the Fc region; second, it binds to C1r and C1s, which are two serine proteases, thereby initiating the formation of the C1 complex.

The CDC activity or ADCC activity of the bispecific antibody of the present invention or the antibody fragment on antigen-expressing cells can be evaluated by a known assay method [Cancer Immunology, Immunotherapy, 36, 373 (1993)].

As a method for controlling the effector activity of the bispecific antibody of the present invention, there are known methods such as controlling fucose (also referred to as N-acetylglucosamine (GlcNAc) bound to the α-1,6 positions The amount of core fucose), the above-mentioned N-acetylglucosamine is present in combination with asparagine (Asn) at position 297 of the Fc region of the antibody (including the constant region of the CH2 domain and the CH3 domain). The reducing end of N-bound complex sugar chain (International Publication No. 2005/035586, International Publication No. 2002/31140, and International Publication No. 2000/61739); or by the amino acid residue of the Fc region of the antibody. Modified and controlled (International Publication No. 2000/42072).

By controlling the amount of fucose added to the bispecific antibody, the ADCC activity of the antibody can be enhanced or decreased. For example, as a method of reducing the content of fucose bound to the N-binding complex sugar chain bound to the Fc of an antibody, a host cell in which the α1,6-fucosyltransferase gene has been deleted is used to express bispecificity antibodies, whereby bispecific antibodies with higher ADCC can be obtained. On the other hand, as a method for increasing the content of fucose bound to the N-binding complex sugar chain bound to the Fc of the bispecific antibody, a host into which an α1,6-fucosyltransferase gene has been introduced is used. cells to express antibodies, whereby bispecific antibodies with lower ADCC activity can be obtained.

Also, by modifying the amino acid residues in the Fc region of the bispecific antibody, ADCC activity or CDC activity can be enhanced or reduced. For example, the CDC activity of the bispecific antibody can be enhanced by using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/0148165. Furthermore, ADCC activity or CDC activity can be enhanced or reduced by carrying out amino acid modification as described in US Pat. No. 6,737,056, US Pat. No. 7,297,775, or US Pat. No. 7,317,091.

Furthermore, bispecific antibodies with controllable effector activity can also be obtained by combining the above methods.

The stability of the bispecific antibodies of the present invention can be assessed by measuring the amount of aggregates (oligomers) formed in a sample stored during the purification process or under certain conditions. That is, when the amount of aggregates decreased under the same conditions, it was evaluated that the stability of the antibody was improved. The amount of aggregates can be determined by separating agglutinated antibody from non-agglutinated antibody using a suitable chromatography method including gel filtration chromatography.

The productivity of the bispecific antibody of the present invention can be evaluated by measuring the amount of antibody produced from the antibody-producing cells in the culture medium. More specifically, it can be evaluated by measuring the amount of the antibody contained in the culture supernatant after removing the antibody-producing cells from the culture medium by an appropriate method such as an HPLC method or an ELISA method.

In the present invention, the term "antibody fragment" refers to a protein containing an antigen-binding site and having antigen-binding activity to the antigen. For example, Fab, Fab', F(ab') ₂ , scFv (single-chain variable fragment, single-chain variable fragment), diabody, dsFv (Disulfide-stabilized Fv, disulfide-stabilized Fv) Fv), VHH (variable domain of heavy chain of heavy-chain antibody, heavy chain single-domain antibody) or peptides containing CDR (Complementarity determining regions, complementarity determining regions), etc.

Fab is a fragment obtained by treating IgG antibody with the proteolytic enzyme papain (cleaved at the amino acid residue at position 224 of the H chain) about half of the N-terminal side of the H chain and the entire L chain through disulfide An antibody fragment with antigen-binding activity with a molecular weight of about 50,000 formed by bonding (S-S bond). The heavy chain of the Fab comprising VH and CH1 was designated as VH-CH1. In addition, the light chain of the Fab containing VL and CL is denoted as VL-CL.

F(ab') ₂ is formed by combining the Fab (cleaved at the amino acid residue at the 234th position of the H chain) in the fragment obtained by treating IgG with the proteolytic enzyme pepsin through the SS bond of the hinge region. A slightly larger antibody fragment with a molecular weight of about 100,000 with antigen-binding activity.

Fab' is an antibody fragment having an antigen-binding activity with a molecular weight of about 50,000 obtained by cleaving the SS bond in the hinge region of the above-mentioned F(ab') ₂ .

scFv is an antibody fragment in the form of a VH-P-VL or VL-P-VH polypeptide with antigen-binding activity, which uses a suitable peptide linker (P) of more than 12 residues to link one VH and one VL. .

Diabodies are antibody fragments with bivalent antigen-binding activity against the same antigen, or with specific antigen-binding activities against different antigens, obtained by dimerizing scFvs with the same or different antigen-binding specificities.

dsFv is a polypeptide in which one amino acid residue in VH and VL is replaced by a cysteine residue through S-S bonds between the cysteine residues.

VHH (also known as nanobody) refers to the variable region of the heavy chain in VHH antibodies that can bind to antigen in the absence of other polypeptides.

VHH antibodies are antibodies that exist in camelid animals such as alpacas and cartilaginous fish such as sharks. They do not contain light chains and CH1, but only heavy chains.

The CDR-containing peptide is composed of at least one region of the VH or VL CDR. Peptides comprising a plurality of CDRs can be made by binding the CDRs directly to each other or via a suitable peptide linker. Peptides comprising CDRs can be constructed by constructing DNA encoding the CDRs of VH and VL of the bispecific antibody of the present invention, inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into Manufactured for expression in prokaryotes or eukaryotes. In addition, peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.

In the present invention, the so-called bispecific antibody fragment refers to a fragment that essentially includes a partial structure of a bispecific antibody and has antigen-binding activity against two antigens.

The present invention also includes: fusion proteins formed by binding the Fc of the bispecific antibody of the present invention to antibody fragments, and Fc fusion proteins formed by binding the Fc to naturally occurring ligands or receptors (also known as immunoadhesins) , and Fc fusion proteins formed by the fusion of multiple Fc regions. In addition, the bispecific antibody of the present invention can also be used in an Fc region containing amino acid residue modifications aimed at enhancing or attenuating the effector activity of the antibody, maintaining antibody stability, and controlling half-life in blood.

As the bispecific antibody or the bispecific antibody fragment of the present invention, it includes chemically or genetically engineering the bispecific antibody or the bispecific antibody fragment of the present invention with radioisotopes, low-molecular-weight drugs, macromolecules Derivatives of antibodies that are combined with drugs, proteins, or antibody drugs.

Derivatives of the antibodies of the present invention can be prepared by chemical methods ["Introduction to Antibody Engineering", Diren Library (1994)], in the H chain or L chain of the bispecific antibodies of the present invention or the bispecific antibody fragments. The N-terminal side or C-terminal side of the chain, a suitable substituent or side chain in the antibody or its antibody fragment, and a sugar chain in the antibody or its antibody fragment, etc., are bound to radioisotopes, low-molecular-weight drugs, and high-molecular-weight drugs. , immune activator, protein or antibody medicine, etc.

In addition, the derivatives of the antibodies of the present invention can be inserted by linking the DNA encoding the bispecific antibody or the bispecific antibody fragment of the present invention with the DNA encoding the desired protein or antibody medicine by genetic engineering. to an expression vector, and then the expression vector is introduced into a suitable host cell for expression and production.

As a radioisotope, for example, ¹¹¹ In, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ⁶⁴ Cu, ⁹⁹ Tc, ⁷⁷ Lu, or ²¹¹ At, etc. may be mentioned. The radioisotope can be directly bound to the antibody by the chloramine T method or the like. In addition, a substance that chelates a radioisotope can also be bound to the antibody. As a chelating agent, 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) etc. are mentioned, for example.

Examples of low-molecular-weight drugs include alkylating agents, nitrosoureas, metabolic antagonists, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, Anticancer agents such as P-glycoprotein inhibitors, platinum complex derivatives, M-phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy Society (1996)], hydrocortisone or prednisone (Prednisone), non-steroidal agents such as aspirin or indomethacin, immunomodulators such as gold thiomalate or penicillamine, immunosuppressants such as cyclophosphamide or azathioprine, or chlorine maleate Anti-inflammatory agents such as antihistamines such as pheniramine or clemastine ["Inflammation and Anti-Inflammation Therapy", Medical & Dental Publishing Co., Ltd. (1982)], etc.

Examples of anticancer agents include amiphodine (Ethyol), cisplatin, dacarbenol (DTIC), actinomycin, dichloromethyldiethylamine (nitrogen mustard), streptozotocin, Cyclophosphamide, Ifosfamide, Carmustine (BCNU), Lomustine (CCNU), Doxorubicin (Adriomycin), Epirubicin, Gemcitabine (Gemcitabine Hydrochloride), Daunorubicin, Procarbazine, Mitomycin, Cytarabine, Etoposide, 5-Fluorouracil, Fluorouracil, Vinblastine, Vincristine, Bleomycin, Daunomycin, Pelor Mycin, estramustine, paclitaxel (Texol), European paclitaxel (Ke cancer easy), aldesleukin, asparaginase, busulfan, carboplatin, oxaliplatin (Oxaliplatin), nedaplatin , cladribine, camptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxy urea, adamycin, mesna, Irinotecan (CPT-11), topotecan, mitoxantrone, topotecan, saupeylin, megestrol, melphalan, mercaptopurine, hydroxy carbamide, fucomacin, Turtan, Peaspargase, Pentostatin, Pipobromide, Streptozotocin, Tamoxifen, Goserelin, Leuprolide, Flutamide, Teniposide, Intratestis Lipid, Thioguanine, Thiatepa, Uracil, Vinorelbine, Chlormustine, Hydrocortisone, Prednisolone, Methylprednisolone, Vindesine, Nimustine , Semustine, Capecitabine, Tomudex, Azacitidine, UFT, Oxaloplatin, Gefitinib (Iressa), Imatinib (STI571), Erlotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitor, vascular endothelial growth factor receptor (VEGFR) inhibitor, fibroblast growth factor receptor (FGFR) inhibitor, Tarceva and other epidermis Growth Factor Receptor (EGFR) Inhibitor, Rhizobactin, 17-Allylamino-17-Demethoxygeldanamycin, Rapamycin, Amacridine, All-Trans Retinoid acid, thalidomide, lenalidomide, anastrozole, fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine, imidazole Ribine, Cyclosporine, Rapamycin, Hydrocortisone, Bexarotene (Targretin), Tamoxifen, Dexamethasone, Progestins, Estrogen, Adderall Natrozole (Arimidex), Leuplin, Aspirin, Indomethacin, Celecoxib, Azathioprine, Penicillamine, Gold Thiomalate, Chlorobenzene Maleate Namin, chlorpheniramine maleate, clemastine, retinoic acid, bexarotene, arsenic, bortezomib, allopurinol, calicheamicin, tiimumab (tisutan) Ibritumomab Tiuxetan), Targretin, Ozogamin , clarithromycin, leucovorin, ketoconazole, aminoglutamine, suramin, methotrexate or maytansinoids or derivatives thereof, etc.

As a method for binding a low-molecular-weight drug to the bispecific antibody of the present invention or the bispecific antibody fragment, for example, a method of binding the drug to the amine group of the antibody via glutaraldehyde, or a A method of binding the amine group of the drug to the carboxyl group of the antibody with carbodiimide, etc.

As the polymer drug, polyethylene glycol (PEG), albumin, dextran, polyoxyethylene, styrene-maleic acid copolymer, polyvinylpyrrolidone, piperane copolymer, or Hydroxypropyl methacrylamide, etc. By combining these high molecular compounds with the bispecific antibody or antibody fragment of the present invention, it can be expected that 1) the stability against various chemical, physical or biological factors is improved, 2) the half-life in blood is significantly prolonged, or 3) Loss of immunogenicity or inhibition of antibody production ["Conjugate Medicines", Guangchuan Bookstore (1993)].

For example, as a method of binding PEG to the bispecific antibody of the present invention, a method of reacting with a PEGylation modification reagent, etc. ["Conjugated Drugs", Hirokawa Shoten (1993)] can be exemplified. Examples of PEGylation modification reagents include: modifiers for the ε-amino group of p-lysine (Japanese Patent Laid-Open No. 61-178926 ), modifiers for the carboxyl groups of p-aspartic acid and glutamic acid ( Japanese Patent Laid-Open No. 56-23587), or a modifier for the guanidine group of arginine (Japanese Patent Laid-Open No. 2-117920).

The immune activator may be a known natural immune adjuvant, and specific examples thereof include immunostimulatory agents: β(1→3) glucan (eg, lentinan or schizophyllan), or α-galactoside Ceramide (KRN7000) and so on.

Examples of the protein include cytokines, growth factors, and toxin proteins that activate immunocompetent cells such as NK cells, macrophages, and neutrophils.

Examples of cytokines and growth factors include interferon (hereinafter referred to as IFN)-α, IFN-β, IFN-γ, interleukin (hereinafter referred to as IL)-2, IL-12, IL-15 , IL-18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (M-CSF) Wait.

Examples of toxin proteins include ricin, diphtheria toxin, ONTAK, and the like, and also include protein toxins in which mutations are introduced into proteins in order to regulate toxicity.

Fusion antibodies to proteins or antibody pharmaceuticals can be constructed by linking the cDNA encoding the bispecific antibody or antibody fragment of the present invention with the cDNA encoding the protein to construct DNA encoding the fusion antibody, and insert the DNA into prokaryotes or genotypes. An expression vector for nuclear organisms is produced by introducing the expression vector into prokaryotes or eukaryotes for expression.

When a derivative of the above-mentioned antibody is used as a detection method, a quantitative method, a detection reagent, a quantitative reagent or a diagnostic drug, as a drug that binds to the bispecific antibody or antibody fragment thereof of the present invention, the usual Labels used in immunological detection methods or assays. Examples of the label include enzymes such as alkaline phosphatase, peroxidase, and luciferase, luminescent substances such as acridinium ester and rophene, or luciferin isothiocyanate (FITC) or isothiocyanate. Fluorescent substances such as tetramethylrhodamine cyanate (RITC), Alexa (registered trademark) Fluor 488, R-kaixin (R-PE), and the like.

The present invention includes bispecific antibodies having cytotoxic activity such as CDC activity or ADCC activity, and bispecific antibody fragments. The CDC activity or ADCC activity of the bispecific antibody of the present invention or the bispecific antibody fragment on antigen-expressing cells can be evaluated by a known assay method ["Cancer Immunology, Immunotherapy", 36, 373 (1993)] .

Furthermore, the present invention relates to a composition comprising a bispecific antibody or a fragment of the bispecific antibody that specifically recognizes and binds to TfR and GPC3, or a composition comprising the bispecific antibody or the fragment of the bispecific antibody as an active ingredient Diseases related to at least one of TfR and GPC3 are preferably therapeutic drugs for diseases in which expressing cells of TfR and GPC3 are involved.

The disease related to at least one of TfR and GPC3 may be any disease related to at least one of TfR and GPC3, and examples thereof include malignant tumors and cancer.

In the present invention, examples of malignant tumors and cancers include colorectal cancer, colorectal cancer, lung cancer, breast cancer, glioma, melanoma, thyroid cancer, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, esophagus cancer, liver cancer, head and neck cancer, skin cancer, urinary tract cancer, bladder cancer, prostate cancer, choriocarcinoma, pharyngeal cancer Carcinoma, laryngeal cancer, mesothelioma, pleural tumor, androblastoma, endometrial hyperplasia, endometriosis, embryonal histoma, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangiomas cell tumor, retinoblastoma, astrocytoma, neurofibroma, oligodendritic cell tumor, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteogenic Sarcoma, leiomyosarcoma and Wilms tumor.

The therapeutic drug containing the bispecific antibody of the present invention or the bispecific antibody fragment or the derivative thereof may contain only the bispecific antibody or the bispecific antibody fragment or the derivative thereof as an active ingredient Usually, it is preferable to provide it in the form of a pharmaceutical preparation which is mixed with one or more pharmacologically acceptable carriers and produced by any method known in the technical field of pharmaceutical preparations.

The route of administration is preferably the one that is most effective in the treatment, for example, oral administration, or parenteral administration such as intraoral, intratracheal, intrarectal, subcutaneous, intramuscular, or intravenous administration. Among them, intravenous administration is preferred.

Examples of the administration form include sprays, capsules, lozenges, powders, granules, syrups, emulsions, suppositories, injections, ointments, and patches.

The dosage or frequency of administration varies according to the target therapeutic effect, administration method, length of treatment course, age and body weight, etc. Generally, the average adult is 10 μg/kg to 10 mg/kg per day.

Furthermore, the present invention relates to a reagent for immunological detection or measurement comprising at least one of TfR and GPC3 of the bispecific antibody or the bispecific antibody fragment of the present invention, or a disease related to at least one of TfR and GPC3 It is preferably a diagnostic drug for diseases in which TfR and GPC3 expressing cells are involved. In addition, the present invention relates to a method for immunological detection or measurement using the bispecific antibody of the present invention or at least one of TfR and GPC3 of the bispecific antibody fragment, and a comparison of diseases related to at least one of TfR and GPC3. Preferably, a method for treating a disease involving TfR and GPC3 expressing cells, or a disease related to at least one of TfR and GPC3 is preferably a method for diagnosing a disease involving expressing cells of TfR and GPC3.

In the present invention, as a method for detecting or measuring the amount of at least one of TfR and GPC3, any known method can be exemplified. For example, an immunological detection or measurement method etc. are mentioned.

The so-called immunological detection or assay method refers to a method for detecting or measuring the amount of antibody or antigen by using labeled antigen or antibody. Examples of immunological detection or measurement methods include radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, Western Ink dot method or physical chemical method, etc.

By detecting or measuring cells expressing at least one of TfR and GPC3 using the bispecific antibody or the bispecific antibody fragment of the present invention, a disease associated with at least one of TfR and GPC3 can be diagnosed, preferably TfR and GPC3 Diseases in which cells are involved.

The detection of cells expressing at least one of TfR and GPC3 can be performed by well-known immunological detection methods, such as immunoprecipitation, immune cell staining, immunohistochemical staining, or fluorescent antibody staining. In addition, fluorescent antibody staining methods such as FMAT8100HTS system (manufactured by Applied Biosystems) and the like can also be exemplified.

In the present invention, as a biological sample to be used for detection or measurement of at least one of TfR and GPC3, as long as it is tissue cells, blood, plasma, serum, body fluid, urine, feces, tissue fluid or culture fluid, it may contain The cells of at least one of TfR and GPC3 are not particularly limited.

The diagnostic drug containing the bispecific antibody of the present invention, the bispecific antibody fragment, or the derivative thereof may also contain a reagent for carrying out an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method. As a reagent for carrying out an antigen-antibody reaction, a buffer, a salt, etc. are mentioned, for example.

As the detection reagent, for example, a labeled secondary antibody bound to the above-mentioned bispecific antibody or the bispecific antibody fragment or the derivative thereof, or a substrate corresponding to the label, etc., are commonly used in immunological detection. or reagents used in the assay.

Hereinafter, the method for producing the bispecific antibody of the present invention, the method for evaluating the activity of the bispecific antibody or the bispecific antibody fragment, and the diseases using the bispecific antibody or the bispecific antibody fragment are specifically described. Methods of treatment and diagnosis.

1. Production method of monoclonal antibody The production method of the monoclonal antibody of the present invention includes the following steps. That is, (1) at least one operation is performed to purify the antigen used as an immunogen and to prepare cells that overexpress the antigen on the cell surface; (2) after immunizing the animal with the antigen, blood is collected, and its antibody titer is detected to determine the extraction of the spleen. (3) preparation of myeloma cells (myeloma); (4) cell fusion of antibody-producing cells and myeloma; (5) screening of fusion tumor groups producing the target antibody; (6) Isolate (separate) monoclonal cells from the fusion tumor population; (7) cultivate fusion tumors for mass production of monoclonal antibodies, or breed animals transplanted with fusion tumors, as appropriate; (8) detect the monoclonal cells thus produced Physiological activity of antibodies and their antigen-binding specificity, or their properties as labeling reagents, etc.

Hereinafter, based on the above-mentioned steps, the method for producing a TfR-binding monoclonal antibody and a GPC3-binding monoclonal antibody for producing a TfR- and GPC3-binding bispecific antibody in the present invention will be described in detail. The method for producing the antibody is not limited to this, and for example, antibody-producing cells other than spleen cells and myeloma can also be used.

(1) Purification of antigen Cells expressing TfR or GPC3 can be obtained by introducing an expression vector comprising a cDNA encoding full or partial TfR or GPC3 into Escherichia coli, yeast, insect cells or animal cells and the like. In addition, at least one of TfR and GPC3 can be purified from various human tumor cultured cells or human tissues expressing at least one of TfR and GPC3 in large quantities, and used as an antigen. In addition, the tumor-cultured cells, the tissue, or the like can also be used directly as an antigen. Furthermore, synthetic peptides containing a partial sequence of TfR or GPC3 can be prepared by chemical synthesis methods such as the Fmoc method or the tBoc method and used for antigens.

TfR or GPC3 used in the present invention can be obtained by using "Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press (1989) or "Molecular Biology Experiments", John Wiley International Publishing Company ( 1987-1997), etc., for example, by the following method, the DNA encoding TfR or GPC3 is expressed in a host cell and produced.

Recombinant vectors are made by inserting a full-length cDNA comprising a portion encoding TfR or GPC3 downstream of the promoter of a suitable expression vector. In place of the above-mentioned full-length cDNA, a DNA fragment of suitable length prepared based on the full-length cDNA and comprising the portion encoding the polypeptide can also be used. Next, the obtained recombinant vector is introduced into a host cell suitable for the expression vector, thereby obtaining a transformant producing TfR or GPC3.

As the expression vector, any one capable of autonomous replication or chromosomal integration in the host cell used and containing a suitable promoter at the position where the DNA encoding TfR or GPC3 can be transcribed can be used.

As the host cell, for example, microorganisms belonging to the genus Escherichia such as Escherichia coli, yeast, insect cells, or animal cells can be used which can express the target gene.

When prokaryotes such as Escherichia coli are used as host cells, the recombinant vector is preferably capable of autonomous replication in prokaryotes, and contains a promoter, a ribosome binding sequence, a DNA comprising a portion encoding TfR or GPC3, and a transcription termination sequence the carrier. In addition, the recombinant vector does not necessarily have a transcription termination sequence, but it is preferable to arrange the transcription termination sequence directly below the structural gene. Furthermore, the recombinant vector may also contain a gene controlling the promoter.

As the recombinant vector, it is preferable to use a plasmid in which the Shine-Dalgarno sequence, which is a ribosome-binding sequence, is adjusted to an appropriate distance (for example, 6 to 18 bases) from the initiation codon. .

In addition, the base sequence of the DNA encoding TfR or GPC3 can be substituted by bases in a manner that is most suitable for codon expression in the host, whereby the productivity of the target TfR or GPC3 can be improved.

As the expression vector, any one capable of functioning in the host cell to be used can be used, and examples thereof include pBTrp2, pBTac1, pBTac2 (manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Pharmacia). Invitrogen Corporation), pGEMEX-1 (Promega Corporation), pQE-8 (Qiagen Corporation), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 ["Agricultural Biological Chemistry", 48, 669(1984)], pLSA1 [Agricultural Biochemistry, 53, 277(1989)], pGEL1 [Proceedings of the National Academy of Sciences, 82, 4306(1985)], pBluescript II SK(-) (Stratagene Company), pTrs30 [prepared by Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared by Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared by Escherichia coli IGHA2 (FERM BP-400), Japan Patent Laid-Open No. 60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Patent Laid-Open No. 60-221091], pTerm2 (US Patent No. 4,686,191 Specification, US Patent No. 4,939,094 specification, specification of U.S. Patent No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system ( Novagen Corporation) or pME18SFL3 (Toyobo Corporation) and so on.

As the promoter, any one capable of functioning in the host cell used can be used. For example, promoters derived from Escherichia coli, bacteriophage, etc., such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, or T7 promoter, can be mentioned. In addition, for example, a tandem promoter formed by connecting two Ptrps in series, a tac promoter, a lacT7 promoter, or a let I promoter or other artificially designed and modified promoters, etc., can also be exemplified.

As the host cell, for example, E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, or E. coli DH5α Wait.

As a method for introducing a recombinant vector into a host cell, any method for introducing DNA into the host cell to be used can be adopted, for example, a method using calcium ions [Proceedings of the National Academy of Sciences of the United States of America, 69, 2110 (1972), Genes, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

When an animal cell is used as a host, any one that can function in animal cells can be used as the expression vector, for example, pcDNAI (manufactured by Invitrogen), pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Patent Japanese Patent Laid-Open No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329 , 840 (1987)], pcDNAI/Amp (manufactured by Invitrogen), pcDNA3.1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 ["J. Biochemistry", 101, 1307 (1987) )], pAGE210, pME18SFL3, pCI (manufactured by Promega), or pKANTEX93 (International Publication No. 97/10354).

As the promoter, any one that can function in animal cells can be used, for example, the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the early promoter of SV40, and the promoter of retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter, or the promoter or enhancer of Moloney mouse leukemia virus. In addition, the enhancer and promoter of the IE gene of human CMV may be used together.

Examples of host cells include human Birch's lymphoma cell Namalwa, African green monkey kidney-derived cell COS, Chinese hamster ovary-derived cell CHO, or human leukemia cell HBT5637 (Japanese Patent Laid-Open No. Sho 63- Bulletin No. 000299) and so on.

As a method for introducing a recombinant vector into a host cell, any method for introducing DNA into animal cells can be used, for example, electroporation method ["Cell Technology", 3, 133 (1990)], calcium phosphate method ( Japanese Patent Laid-Open No. Hei 2-227075), or the lipofection method [Proceedings of the National Academy of Sciences of the United States of America, 84, 7413 (1987)], etc.

The transformants derived from microorganisms or animal cells, etc. obtained by the above-mentioned method and possessing the recombinant vector that integrates the DNA encoding TfR or GPC3 are cultivated in a medium, so that at least one of the TfR and GPC3 is accumulated in the culture, from The culture is harvested whereby TfR or GPC3 can be produced. The method of culturing the transformant in the medium can be carried out according to the method generally used for host culture.

In the case of expression in cells derived from eukaryotes, sugar- or sugar-chain-attached TfR or GPC3 can be obtained.

When the microorganism is transformed with a recombinant vector using an inducible promoter and then cultured, an inducer may be added to the medium if necessary. For example: in the case of culturing the microorganism transformed with the recombinant vector using the lac promoter, isopropyl-β-D-thiogalactofuranoside, etc. can be added to the medium; when culturing the microorganism using the trp promoter In the case of the microorganism transformed by the recombinant vector, indole acrylic acid and the like can be added to the culture medium.

As a medium for culturing a transformant obtained by using animal cells as a host, for example, the commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Society for Experimental Biology and Medicine Journal (Proc. Soc. Exp. Biol. Med.)", 73, 1 (1950)], Iscove's modified Dulbecco's medium (IMDM) medium, or a medium supplemented with fetal bovine serum (FBS), etc. . Culture is usually carried out for 1 to 7 days under conditions such as pH 6-8, 30-40°C, and 5% CO ₂ . In addition, antibiotics such as kanamycin and penicillin may be added to the culture medium if necessary during the culture.

As the expression method of the gene encoding TfR or GPC3, in addition to direct expression, methods such as secretory production or fusion protein expression can also be used ["Molecular Colonization: Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press (1989) )]. As a production method of TfR such as EGFR or GPC3, for example, a method of producing it in a host cell, a method of secreting it to the outside of the host cell, or a method of producing it on the outer membrane of the host cell can be exemplified depending on the host cell to be used or the method of producing it. The appropriate method is selected according to the structure of the produced TfR or GPC3.

For example, the DNA encoding the amino acid sequence of the extracellular region is prepared with DNA encoding the Fc region of an antibody, DNA encoding glutathione S-transferase (GST), or DNA encoding a FLAG tag or DNA encoding a Histidine tag. An antigen fusion protein can be produced by expressing and purifying DNA obtained by linking DNA or the like. Specifically, for example, an Fc fusion protein in which the extracellular region of TfR or GPC3 is bound to the Fc region of human IgG, and the extracellular region of TfR or GPC3 and glutathione S-transferase (GST) can be exemplified. fusion protein.

In the case of producing TfR or GPC3 in the host cell or on the outer membrane of the host cell, by using the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], Lowe et al. The method of et al. ["Proceedings of the National Academy of Sciences of the United States of America", 86, 8227 (1989), "Genes Develop.", 4, 1288 (1990)], Japanese Patent Laid-Open No. 05-336963 or International Publication According to the method described in No. 94/23021, etc., TfR or GPC3 can be actively secreted outside the host cell. In addition, a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Laid-Open No. 2-227075 ) can also be used to increase the production amount of TfR or GPC3.

The produced TfR or GPC3 can be isolated and purified, for example, in the following manner.

When TfR or GPC3 is expressed in a dissolved state in the cells, the cells are recovered by centrifugation after culturing, suspended in an aqueous buffer, and then use an ultrasonic crusher, a French crusher, a Manton Gaulin homogenizer or a DYNO ball mill. The cells were disrupted to obtain a cell-free extract. The cell-free extract is centrifuged, and the supernatant is obtained from the obtained supernatant, by single or combined use of the usual protein isolation and purification methods, namely solvent extraction, salting out method using ammonium sulfate, desalting method, using organic Solvent precipitation, anion exchange chromatography using resin such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), using S-Sepharose FF (manufactured by Pharmacia) Cation exchange chromatography using resins such as butyl sepharose, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatographic focusing, The purified protein can be obtained by electrophoresis such as isoelectric point electrophoresis.

When TfR or GPC3 is expressed as an insoluble body in the cells, the cells are collected, disrupted, and centrifuged in the same manner as above to collect the insoluble body of TfR or GPC3 as a precipitate. The recovered insoluble TfR or GPC3 was made soluble using a protein modifier. The solubilized solution is diluted or dialyzed to restore the normal three-dimensional structure of TfR or GPC3, and the purified protein of the polypeptide can be obtained by the same isolation and purification method as above.

In the case of extracellular secretion of TfR or GPC3 or derivatives of these sugar-modified products, the derivatives of TfR or GPC3 or these sugar-modified products can be recovered from the culture supernatant. Purified protein can be obtained from the soluble fraction by treating the culture supernatant by centrifugation or the like in the same manner as described above to obtain a soluble fraction, and using the same isolation purification method as described above.

In addition, in the present invention, TfR or GPC3 to be used may be produced by chemical synthesis methods such as the Fmoc method or the tBoc method. Specifically, chemical synthesis can be performed using, for example, a peptide synthesizer manufactured by Advanced Chemtec, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, Perceptive, or Shimadzu Corporation.

(2) Preparation steps of antibody-producing cells Animals such as mice, rats, hamsters, rabbits, cows, or camels are immunized with the antigen obtained in (1), and antibody-producing cells in the spleen, lymph nodes or peripheral blood of the animals are collected. Further, examples of animals include: gene-transformed mice producing human antibodies described in Tomizuka et al. [Tomizuka et al., Proceedings of the National Academy of Sciences, 97, 722 (2000)], The immunized animals, such as TfR or GPC3 conditional knockout mice, are used to improve immunogenicity.

Immunization is performed by administering the antigen with a suitable adjuvant such as complete Freund's adjuvant, or aluminum hydroxide gel and pertussis vaccine. The immunogen administration method during mouse immunization can be any method such as subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection or plantar injection, preferably intraperitoneal injection, plantar injection or intravenous injection Internal injection. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA (Bovine Serum Albumin) or KLH (Keyhole Hemocyanin) is prepared and used as an immunogen.

After the first antigen administration, another 5 to 10 administrations were performed, with an interval of 1 to 2 weeks between each time. Blood was collected from the fundus venous plexus on the 3rd to 7th day after each administration, and the serum antibody titer was determined by enzyme immunoassay ["Antibody: Laboratory Manual", Cold Spring Harbor Laboratory (1988)]. If the above-mentioned sera show sufficient antibody titers to the antigen used for immunization as a supply source of antibody-producing cells for fusion, the effect of subsequent operations can be improved.

On days 3 to 7 after the last administration of the antigen, tissue containing antibody-producing cells such as spleen was excised from the immunized animal, and antibody-producing cells were collected. Antibody-producing cells are plasma cells and lymphocytes as their precursor cells, which can be obtained from any part of the individual, generally from the spleen, lymph nodes, bone marrow, tonsils, peripheral blood, or an appropriate combination of parts, etc. The most commonly used are spleen cells. When spleen cells are used, the spleen is crushed and dispersed, followed by centrifugation to further remove red blood cells, thereby obtaining fusion antibody-producing cells.

(3) Preparation steps of myeloma As myeloma, cells derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits or humans that do not have the ability to produce antibodies can be used, and cultured cells obtained from mice are generally used. Cell lines, such as 8-azaguanine-resistant mouse (derived from BALB/c) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology )", 18, 1 (1978)], P3-NS1/1-Ag41(NS-1) [European J. Immunology, 6, 511 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653(653) [J. Immunology, 123, 1548 (1979)], or P3-X63 -Ag8(X63) [Nature, 256, 495 (1975)], etc. The cells were grown in a suitable medium such as 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FCS and 8-azaguanine], Iscove's modified Dulbecco medium (hereinafter referred to as "IMDM") or Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM") for subculture. 3 to 4 days before cell fusion, the above cell lines were subcultured in normal medium (eg DMEM medium containing 10% FCS) to ensure that the number of cells on the day of fusion was more than 2×10 ⁷ .

(4) Cell fusion The antibody-producing cells for fusion obtained in (2) and the myeloma cells obtained in (3) were mixed with minimal essential medium (MEM) or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, and 7.65 g of common salt). , 1 L of distilled water, pH 7.2) was thoroughly washed, mixed at a ratio of antibody-producing cells for fusion: myeloma cells = 5:1 to 10:1, centrifuged, and the supernatant was removed. After the precipitated cell aggregates were sufficiently dispersed, a mixed solution of polyethylene glycol-1000 (PEG-1000), MEM medium and dimethylsulfite was added at 37°C while stirring. Furthermore, 1 to 2 mL of MEM medium was added every 1 to 2 minutes, and after several additions, the total amount including the MEM medium was 50 mL. After centrifugation, the supernatant was removed, the precipitated cell aggregates were gradually dispersed, and the cells were slowly suspended in HAT medium [normal medium supplemented with hypoxanthine, thymidine and aminopterin]. The suspension was incubated in a 5% CO ₂ incubator at 37°C for 7-14 days.

In addition, cell fusion can also be performed by the following method. The spleen cells and myeloma cells were thoroughly washed with serum-free medium (such as DMEM) or phosphate buffered saline (hereinafter referred to as "phosphate buffer"), and the ratio of spleen cells to myeloma cells was 5:1 After mixing at about 10:1, centrifugation was performed. The supernatant was removed to fully disperse the precipitated cell aggregates, and a serum-free medium containing 1 mL of 50% (w/v) polyethylene glycol (molecular weight 1000-4000) was added dropwise while stirring. After that, 10 mL of serum-free medium was slowly added, followed by centrifugation. The supernatant was removed again, and the precipitated cells were suspended in a normal medium (hereinafter referred to as HAT medium) containing an appropriate amount of hypoxanthine-aminopterin-thymidine (HAT) solution and human interleukin-2 (IL-2). ), it was dispensed into each well of a well plate for culture (hereinafter referred to as well plate), and cultured at 37°C for 2 weeks in the presence of 5% carbon dioxide. The HAT medium was appropriately supplemented halfway through.

(5) Selection of fusion tumor group When the myeloma cells used for fusion are 8-azaguanine-resistant strains, i.e., hypoxanthine-guanine-phosphoribosyltransferase (HGPRT)-deficient strains, the unfused myeloma cells and the myeloma cells interact with each other. Fused cells cannot survive in HAT medium. On the other hand, fused cells of antibody-producing cells and fusions of antibody-producing cells and myeloma cells can survive in the HAT medium, but fused cells of antibody-producing cells die soon. Therefore, by continuously culturing in the HAT medium, only the fusion tumor of the antibody-producing cells and the myeloma cells is left, and as a result, the fusion tumor can be obtained.

For the fusion tumors that gradually grew into colonies, the HAT medium was exchanged for a medium from which aminopterin was removed (hereinafter referred to as HT medium). Then, a part of the culture supernatant was collected, and antibody-producing fusion tumors were selected by the antibody titer measurement method described later. Examples of methods for measuring antibody titers include various known techniques such as radioisotope immunoassay (RIA), solid-phase enzyme immunoassay (ELISA), fluorescent antibody method, and passive hemagglutination. The RIA method or the ELISA method is preferable from the viewpoints of detection sensitivity, rapidity, accuracy, and automation.

The fusion tumor determined to produce the desired antibody after antibody titer determination was transferred to another well plate for colonization. Examples of the colonization method include: a limiting dilution method in which each well of a well plate contains 1 cell, a soft agar method in which colonies are recovered after culturing in a soft agar medium, and isolated by a micromanipulator. Single cell method, single cell isolation method using cell sorter, etc.

For the wells in which the antibody titer was confirmed, colonization, for example, by limiting dilution method, was repeated 2 to 4 times, and those with stably confirmed antibody titers were selected as fusion tumor strains producing monoclonal antibodies against TfR or GPC3.

(6) Preparation of monoclonal antibodies For 8-10-week-old mice or nude mice treated with pristane (intraperitoneal administration of 2,6,10,14-tetramethylpentadecane (Pristane) 0.5 mL, rearing for 2 weeks) The monoclonal antibody obtained by intraperitoneal injection (5) produced a fusion tumor. After 10 to 21 days, the ascites of the fusion tumor became cancerous. The ascites was collected from the mouse, and after centrifugation to remove solid components, salting out with 40-50% ammonium sulfate, and using caprylic acid precipitation, DEAE-Sepharose column, protein A column or gel filtration column Purification and collection of IgG or IgM fractions as purified monoclonal antibodies. In addition, the fusion tumor is propagated in the intraperitoneal cavity of syngeneic mice (such as BALB/c) or Nu/Nu mice, rats, guinea pigs, hamsters, or rabbits, whereby a large amount of TfR or GPC3-binding proteins can be obtained. Monoclonal antibody ascites.

The monoclonal antibody-producing fusion tumor obtained in (5) is cultured in RPMI1640 medium supplemented with 10% FBS, etc., the supernatant is removed by centrifugation, and suspended in GIT medium or Hybridoma SFM medium supplemented with 5% Daigo's GF21 etc., culture by flask culture, spin culture, back culture, etc. for 3 to 7 days. The obtained cell suspension is centrifuged, and the obtained supernatant is purified by protein A column or protein G column, and the IgG fraction is collected, and purified monoclonal antibody can also be obtained. As a simple method for purification, a commercially available monoclonal antibody purification kit (eg, MAbTrap GII kit; manufactured by Amersham Pharmacia Biotech) and the like can also be used.

Antibody subtypes were determined by enzyme immunoassay using a subtype classification kit. The quantification of protein amount was carried out by Lowry's method and a method calculated according to absorbance at ₂₈₀ nm [1.4(OD280)=immunoglobulin 1 mg/mL].

(7) Binding analysis of monoclonal antibody to TfR or GPC3 The binding activity of monoclonal antibody to TfR or GPC3 can be analyzed by Ouchterlony, ELISA, RIA, flow cytometry (FCM) or surface plasmon resonance (SPR) System measurement.

The Euclidean double diffusion method is relatively simple, but concentration operation is required when the antibody concentration is low. On the other hand, in the case of using the ELISA method or the RIA method, the culture supernatant is directly reacted with the antigen-adsorbing solid phase, and antibodies corresponding to various immunoglobulin isotypes and subtypes are used as secondary antibodies. The binding activity of the antibody is determined while identifying the isotype and isotype of the antibody.

As a specific example of the step, purified or partially purified TfR or GPC3 is adsorbed on a solid surface such as a 96-well plate for ELISA, and then the non-adsorbed solid surface is blocked with an antigen-independent protein such as bovine serum albumin (BSA). . The ELISA well plate was washed with phosphate buffered saline (PBS) and PBS containing 0.05% Tween20 (Tween-PBS), etc., followed by serially diluted primary antibodies (such as mouse serum, culture supernatant, etc.) The reaction allows the antibody to bind to the antigen immobilized on the well plate. Next, an anti-immunoglobulin antibody labeled with biotin, an enzyme (horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.), a chemiluminescent substance, or a radioactive compound, etc., is dispensed as the second antibody, so that The second antibody reacts with the first antibody bound to the well plate. After thorough washing with Tween-PBS, a corresponding reaction is performed according to the labeling substance of the second antibody, and a monoclonal antibody that specifically reacts with the target antigen is selected.

The binding activity of an antibody to antigen-expressing cells can be determined by the FCM method [Cancer Immunology, Immunotherapy, 36, 373 (1993)]. Binding of an antibody to a membrane protein antigen expressed on the cell membrane means that the antibody recognizes and binds to the three-dimensional structure of a naturally occurring antigen.

As an SPR method, the kinetics analysis by Biacore can be mentioned. For example, Biacore T100 is used to measure the kinetics of the binding between the antigen and the test substance, and the results are analyzed using the analysis software attached to the machine. As a specific example of the procedure, after immobilizing the anti-mouse IgG antibody on the sensor chip CM5 by the amine coupling method, a test substance such as a fusion tumor culture supernatant or a purified monoclonal antibody is circulated to bind an appropriate amount, and the circulating concentration is known. Binding and dissociation were measured at multiple concentrations of antigen. Secondly, use the software attached to the machine to perform dynamic analysis based on a 1:1 combination model on the obtained data to obtain various parameters. Alternatively, after immobilizing TfR or GPC3 on the sensor chip by, for example, amine coupling, multiple concentrations of purified monoclonal antibodies with known concentrations are passed through to measure binding and dissociation. Using the software attached to the machine, the obtained data is subjected to kinetic analysis based on a bivalent binding model, and various parameters are obtained.

Furthermore, in the present invention, an antibody that competes with an antibody against TfR or GPC3 for binding to TfR or GPC3 can be selected by reacting with the test antibody in the aforementioned binding assay system. That is, by screening an antibody whose binding to the antigen is inhibited when the test antibody is added, an antibody that competes with the antibody obtained above for binding to TfR or GPC3 can be obtained.

(8) Identification of epitopes of monoclonal antibodies against TfR or GPC3 In the present invention, the epitope recognized and bound by the antibody can be identified as follows.

For example, when making a partial deletion of an antigen, a variant obtained by remodeling amino acid residues that differ between species, or a variant obtained by remodeling a specific region, if the antibody responds to the deletion or variant If the resistance is reduced, it indicates that the deletion site or amino acid modification site is the epitope of the antibody. Partial deletions and variants of such antigens can be obtained in the form of secreted proteins using suitable host cells such as Escherichia coli, yeast, plant cells or mammalian cells, or expressed as antigens on the cell membrane of host cells Prepared in the form of cells. In the case of a membrane-type antigen, in order to maintain the three-dimensional structure of the antigen during expression, it is preferably expressed on the membrane of the host cell. In addition, a synthetic peptide imitating the primary structure or the three-dimensional structure of the antigen can also be produced to confirm the reactivity of the antibody. As for the synthetic peptide, a method of producing various partial peptides of the molecule by using a known peptide synthesis technique, etc. may be mentioned.

For example, a chimeric protein can be produced by appropriately combining the domains constituting each region against the extracellular regions of human and mouse TfR or GPC3, and the reactivity of the antibody to the protein can be confirmed, thereby identifying the epitope of the antibody. Afterwards, various oligopeptides corresponding to the above-mentioned proteins, or variants of the peptides are synthesized in more detail by using oligopeptide synthesis techniques well-known in the industry, and the reactivity of the antibody to the peptides is confirmed, whereby epitopes can be specified. As a convenient method for obtaining various oligopeptides, commercially available kits (for example, SPOTs kit (manufactured by Genosys Biotechnologies), a series of multicenter peptide synthesis kits (manufactured by Kayon), etc. ].

An antibody that binds to the same epitope as an antibody that binds to TfR or GPC3 can be obtained by identifying the epitope of the obtained antibody using the above-mentioned binding assay system, and preparing a partial synthetic peptide of the epitope , a synthetic peptide imitating the three-dimensional structure of the epitope, or a recombinant of the epitope, etc., to implement immunization.

For example, if the epitope is a membrane protein, the whole extracellular region or part of the extracellular region is linked with a suitable tag such as FLAG tag, Histidine tag, GST protein or antibody Fc region to make a recombinant fusion protein. Immunization is performed, whereby the epitope-specific antibody can be produced more efficiently.

2. Production of recombinant antibody As an example of the production of recombinant antibodies, in P. J. Delves., "Antibody Production Technology (ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES)", 1997, John Wiley Press (WILEY), P. Shepherd and C. Dean., "Individual Antibodies (Monoclonal Antibodies)", 2000, Oxford University Press, and J. W. Goding., "Principle and Operation of Monoclonal Antibodies", 1993, Academic Press, etc. are outlined, the following exemplifies chimeric antibodies, humanized antibodies and Methods of making human antibodies. In addition, recombinant mouse, rat, hamster and rabbit antibodies can also be produced by the same method.

(1) Acquisition of cDNA encoding the V region of a monoclonal antibody derived from a fusion tumor For example, cDNAs encoding VH and VL of a monoclonal antibody can be obtained in the following manner.

First, mRNA is extracted from a monoclonal antibody-producing fusion tumor, and cDNA is synthesized. Next, the synthesized cDNA is cloned into a vector such as phage or plasmid to prepare a cDNA gene library. Recombinant phages or recombinant plasmids containing cDNA encoding VH or VL, respectively, were isolated from the gene pool using DNA encoding either the C region portion or the V region portion of the antibody as a probe. The complete base sequence of VH or VL in the isolated recombinant phage or recombinant plasmid is determined, and the complete amino acid sequence of VH or VL is deduced from the base sequence.

As a non-human animal for producing a fusion tumor, a mouse, a rat, a hamster, a rabbit, or the like is used, and any animal capable of producing a fusion tumor can be used.

For the preparation of total RNA from a fusion tumor, a kit such as the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] or RNA easy kit (manufactured by Qiagen) is used. Wait.

mRNA preparation from whole RNA using oligo(dT)-immobilized cellulose column method [Molecular Colonization: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)] or oligo dT30 <Super> mRNA purification Kits (Oligo-dT30 <Super> mRNA Purification Kit) (manufactured by Takara Biological Co., Ltd.) and the like. Alternatively, mRNA can be prepared using a kit such as FastTrack mRNA Isolation Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

Well-known methods were used for cDNA synthesis and cDNA gene library preparation ["Molecular Colony: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press (1989), "Molecular Biology Laboratory Manual", Supplement 1) , John Wiley International Publishing Company (1987-1997)] or SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Invitrogen) or ZAP-cDNA synthesis kit (ZAP- cDNA Synthesis Kit) (manufactured by Stratagene), etc.

When preparing a cDNA gene library, as a vector for integrating cDNA synthesized using mRNA extracted from a fusion tumor as a template, any vector capable of integrating the cDNA can be used.

For example, using ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) ["Nucleic Acid Research", 17, 9494 (1989)], λZAPII (manufactured by Stratagene), λgt10, λgt11 ["DNA Colonization") : Practical Methods (DNA Cloning: A Practical Approach), 1, 49 (1985)], Lambda BlueMid (manufactured by Clontech), λExCell, pT7T3-18U (manufactured by Pharmacia), pcD2 ["Molecular Cell Biology (Mol. Cell. Biol.)", 3, 280 (1983)] or pUC18 ["Gene", 33, 103 (1985)] and so on.

Escherichia coli for introduction of a cDNA gene library constructed from a phage or a plasmid vector can be any one capable of introducing, expressing and maintaining the cDNA gene library. For example using XL1-Blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983) ], NM522 [Journal of Molecular Biology, 166, 1 (1983)], K802 [Journal of Molecular Biology, 16, 118 (1966)] or JM105 [Genes, 38, 275 (1985)], etc. .

Colony hybridization or plaque hybridization using isotopically or fluorescently labeled probes is used to select cDNA clones encoding VH or VL of non-human antibodies from a cDNA gene library [Molecular Cloning: A Laboratory Manual , 2nd ed., Cold Spring Harbor Laboratory Press (1989)] et al.

In addition, primers can also be prepared, and the polymerase chain reaction method can be carried out using cDNA synthesized from mRNA or a cDNA gene library as a template [hereinafter referred to as PCR method, "Molecular Colonization: Laboratory Manual", 2nd Edition, Cold Spring Harbor Experiment. Laboratory Press (1989), "Handbook of Molecular Biology", Supplement 1, John Wiley International Publishing Company (1987-1997)], thereby preparing cDNA encoding VH or VL.

The selected cDNA is cut with a suitable restriction enzyme, etc., and cloned into a plasmid such as pBluescript SK(-) (manufactured by Stratagene), and the nucleotide sequence of the cDNA is determined by a commonly used nucleotide sequence analysis method or the like. For example, after performing a reaction such as the dideoxy method [Proceedings of the National Academy of Sciences, 74, 5463 (1977)], analysis is performed using an automatic base sequence analyzer such as A.L.F. DNA Sequencer (manufactured by Pharmacia).

Based on the determined complete base sequences, the complete amino acid sequences of VH and VL were deduced, respectively, and the complete amino acid sequences of VH and VL of known antibodies ["Hot Immunological Protein Sequences", US Department of Health and Human Services (US) Dept. Health and Human Services) (1991)] to confirm whether the obtained cDNA encodes the complete amino acid sequence of each of the VH and VL of the antibody including the secretion signal sequence.

For the complete amino acid sequence of each of the VH and VL of the antibody, including the secretion signal sequence, by comparing the complete amino acid sequence of the VH and VL of the known antibody ["Hot Immunology Protein Sequences", US Health and Human Services (1991)], the length and N-terminal amino acid sequence of the secretion signal sequence can be estimated, and the subgroups to which they belong can be identified.

Also, the amino acid sequences of each CDR of VH and VL were compared with the amino acid sequences of VH and VL of known antibodies ["Hot Immunological Protein Sequences," US Department of Health and Human Services (1991)]. presumption.

Also, regarding the obtained complete amino acid sequences of VH and VL, for example, using any database such as SWISS-PROT or PIR-Protein, a BLAST-based method is performed ["Journal of Molecular Biology," 215, 403 (1990)] Homology searches such as these can confirm whether the complete amino acid sequences of the VH and VL are novel.

(2) Construction of a vector for expression of recombinant antibody The recombinant antibody expression vector can be constructed by breeding DNA encoding at least one of CH and CL of a human antibody in an animal cell expression vector.

As the C region of a human antibody, CH and CL of any human antibody can be used, for example, CH of γ1 subtype and CL of κ type of human antibody can be used. As the DNA encoding CH and CL of the human antibody, cDNA is used, and chromosomal DNA including exons and introns can also be used.

As an expression vector for animal cells, any one capable of integrating and expressing a gene encoding the C region of a human antibody can be used. ", 101, 1307 (1987)], pHSG274 ["Gene", 27, 223 (1984)], pKCR ["Proceedings of the National Academy of Sciences, 78, 1527 (1981)], pSG1bd2-4 ["Cytology ", 4, 173 (1990)] or pSE1UK1Sed1-3 ["Cell Technology", 13, 79 (1993)], INPEP4 (manufactured by Biogen-IDEC), N5KG1val (US Patent No. 6,001,358 specification), N5KG4PE R409K ( International Publication No. 2006/033386), N5KG2 vector (described in International Publication No. 2003/033538), transposon vector (International Publication No. 2010/143698), and the like.

As the promoter and enhancer of the expression vector for animal cells, the early promoter of SV40 [Journal of Biochemistry, 101, 1307 (1987)], Moloney mouse leukemia virus LTR ["Biochemistry and Biophysics] can be used. Research Communications (Biochem. Biophys. Res. Commun.)", 149, 960 (1987)], CMV promoter (US Patent No. 5,168,062 specification) or immunoglobulin H chain promoter ["Cell (Cell)", 41, 479 (1985)] and enhancers [Cell, 33, 717 (1983)] and so on.

From the viewpoints of the ease of construction of the vector, the ease of introduction into animal cells, and the balance of the expression levels of the H chain and L chain of the intracellular antibody, when expressing the recombinant antibody, the H chain and L chain of the antibody are used. The gene vector (tandem type vector) [J. Immunol. Methods, 167, 271 (1994)], or a vector carrying the antibody H chain gene and a vector carrying the L chain can also be used in combination Plural vectors of gene vectors (separate vectors).

As vectors for expression of tandem-type recombinant antibodies, pKANTEX93 (International Publication No. 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], N5KG1val (US Patent No. 6,001,358 specification), N5KG4PE R409K (International Publication No. 1998) were used No. 2006/033386), N5KG2 vector (described in International Publication No. 2003/033538), Tol2 transposon vector (described in International Publication No. 2010/143698), and the like.

(3) Construction of chimeric antibody expression vector The cDNA encoding the VH or VL of the non-human antibody obtained in (1) is cloned upstream of each of the genes encoding the CH or CL of the human antibody in the vector for expression of the recombinant antibody obtained in (2), whereby a chimeric chimera can be constructed. A carrier for antibody expression.

First, in order to link the 3' end side of the cDNA encoding the VH or VL of the non-human antibody to the 5' end side of the CH or CL of the human antibody, the nucleotide sequence of the linking moiety encodes an appropriate amino acid, and The cDNAs of VH and VL were designed in such a way as to be suitable restriction enzyme recognition sequences. Next, the prepared VH and VL cDNAs are cloned respectively upstream of the genes encoding the CH or CL of the human antibody in the vector for expression of the recombinant antibody obtained in (2) to express them in a suitable form, and then construct A vector for chimeric antibody expression.

In addition, synthetic DNA containing appropriate restriction enzyme recognition sequences at both ends can also be used to amplify the cDNA encoding the VH or VL of the non-human antibody by PCR method, and colonize the gene recombinant antibody expression vector obtained in (2) , thereby constructing a chimeric antibody expression vector.

(4) Preparation of cDNA encoding the V region of a humanized antibody The cDNA encoding the VH or VL of the humanized antibody can be prepared as follows. First, the amino acid sequence of the framework region (hereinafter referred to as FR) of the VH or VL of the human antibody to which the amino acid sequence of the CDR of the VH or VL of the non-human antibody obtained in (1) was transplanted was selected, respectively.

The amino acid sequence of the selected FR can be any one derived from a human antibody. For example, use the amino acid sequences of FRs of human antibodies registered in databases such as Protein Data Bank, or the common amino acid sequences of FR subgroups of human antibodies ["Hot Immunology Protein Sequences", vol. US Department of Health and Human Services (1991)] and so on. In order to suppress the decrease in the binding activity of the antibody, the amino acid sequence of the human FR which has the highest possible homology (above 60%) with the amino acid sequence of the FR of the VH or VL of the original non-human antibody is selected.

Next, the amino acid sequences of the CDRs of the original non-human antibody are grafted into the amino acid sequences of the FRs of the VH or VL of the selected human antibody, respectively, and the amino acid sequences of the VH or VL of the humanized antibody are designed respectively. . Considering the frequency of codon usage seen in the base sequence of antibody genes ["Hot Immunological Protein Sequences", US Department of Health and Human Services (1991)], the designed amino acid sequence was converted into a DNA sequence, Thereby, the cDNA sequences of VH or VL of the humanized antibody were designed, respectively.

Based on the designed cDNA sequence, several synthetic DNAs with a length of about 100-150 bases were synthesized, and PCR reactions were performed using these. In this case, from the viewpoints of the reaction efficiency of the PCR reaction and the DNA length that can be synthesized, it is preferable to design 4 to 6 synthetic DNAs for the H chain and the L chain, respectively. In addition, synthetic DNA of the full length of the variable region can also be used.

Furthermore, appropriate restriction enzyme recognition sequences are introduced into the 5' ends of the synthetic DNA at both ends, whereby the VH or VL encoding the humanized antibody can be easily cloned in the vector for expression of the recombinant antibody obtained in (2). the cDNA. After the PCR reaction, the amplified products were cloned into plasmids such as pBluescript SK(-) (manufactured by Stratagene), and the nucleotide sequence was determined by the same method as described in (1) to obtain a human containing the desired code. A plasmid containing the DNA sequence of the amino acid sequence of the VH or VL of the antibody.

(5) Modification of the amino acid sequence of the V region of the humanized antibody The antigen-binding activity of a humanized antibody in which only the CDRs of the VH and VL of a non-human antibody are grafted into the FR of the VH and VL of a human antibody is reduced compared to that of the original non-human antibody [BIO/TECHNOLOGY, 9 , 266 (1991)]. Therefore, in the amino acid sequences of the VH and VL FRs of human antibodies, the amino acid residues that directly participate in antigen binding, the amino acid residues that interact with the amino acid residues of the CDRs, and the steric maintenance of the antibody were identified. The amino acid residues indirectly involved in antigen binding are replaced by amino acid residues of the original non-human antibody, so that the antigen binding activity of the reduced humanized antibody can be improved. .

In order to identify the amino acid residues of FRs related to antigen-binding activity, X-ray crystallographic analysis [Journal of Molecular Biology, 112, 535 (1977)] or computer simulation ["Protein Engineering", 7, 1501 (1994)], etc., to carry out the construction and analysis of the three-dimensional structure of the antibody. In addition, several types of modified antibodies are produced for different antibodies, and the relationship between each of them and the antigen-binding activity is repeatedly studied.

The PCR reaction described in (4) was carried out using the synthetic DNA for modification, whereby the amino acid residues of the FR of the VH and VL of a human antibody can be modified. For the amplified product after the PCR reaction, the nucleotide sequence is determined by the method described in (1), and it is confirmed whether the target modification is achieved.

(6) Construction of humanized antibody expression vector The cDNA encoding VH or VL of the constructed humanized antibody is cloned on the upstream of each gene encoding the CH or CL of the human antibody of the recombinant antibody expression vector obtained in (2), and the humanized antibody can be constructed. performance carrier.

For example, by introducing appropriate restriction enzyme recognition sequences into the 5' ends of the synthetic DNAs located at both ends in the synthetic DNAs obtained in (4) and (5) used in constructing the VH or VL of the humanized antibody, respectively, The upstream of each gene encoding the CH or CL of the human antibody in the gene-recombinant antibody expression vector obtained in (2) is cloned, so that these can be expressed in a suitable form.

(7) Construction of a vector for human antibody expression When an animal for producing human antibody is used as an immunized animal to construct a monoclonal antibody-producing fusion tumor, the amino acid sequences and cDNA sequences of VH and VL of the human antibody can be obtained in (1). Here, the gene encoding VH or VL of the human antibody obtained in (1) is cloned upstream of each gene encoding the CH or CL of the human antibody of the vector for expression of the recombinant antibody obtained in (2), thereby constructing Vector for expression of human antibodies.

(8) Transient performance of recombinant antibody Using the vectors for expression of recombinant antibodies obtained in (3), (6) and (7), or expression vectors transformed from these to express recombinant antibodies transiently, a variety of recombinant antibodies obtained can be efficiently evaluated antigen-binding activity.

The host cell into which the expression vector is introduced can be any host cell capable of expressing the gene recombinant antibody, for example, COS-7 cells [American Type Culture Collection (ATCC) number: CRL1651]. The expression vector was introduced into COS-7 cells by DEAE-dextran method ["Methods in Nucleic Acids Res.", CRC Publishing Co., Ltd. (1991)] or lipofection method ["National Academy of Sciences Academic Journal, 84, 7413 (1987)] and so on.

After introducing the expression vector, the enzyme immune antibody method was used ["Principle and Operation of Monoclonal Antibodies", 3rd Edition, Academic Press (1996), "Antibody: Laboratory Manual", Cold Spring Harbor Laboratory (1988), "Monoclonal Antibody" Antibody Experiment Manual", Kodansha Science and Technology (1987)] etc. to measure the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant.

(9) Acquisition of stable expression strains of recombinant antibodies and preparation of recombinant antibodies The recombinant antibody expression vector obtained in (3), (6) and (7) is introduced into a suitable host cell, thereby obtaining a transformant that stably expresses the gene recombinant antibody.

As the introduction of the expression vector into the host cell, for example, the electropenetration method [Japanese Patent Laid-Open No. 2-257891, "Cell Technology", 3, 133 (1990)], the calcium ion method, the electropenetration method can be mentioned. method, spheroplast method, lithium acetate method, calcium phosphate method, lipofection method, etc. In addition, as a method of introducing a gene into an animal described later, for example, a microinjection method, a method of introducing a gene into ES cells by electroporation or lipofection, and a nuclear transfer method can be mentioned.

Any host cell capable of expressing a genetically recombinant antibody can be used as the host cell into which the vector for expression of the genetically recombinant antibody is introduced. For example, mouse SP2/0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), Chinese hamster CHO-K1 cells (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro -5 cells (ATCC CCL-1781), CHO-S cells (Life Technologies, Cat No. 11619), CHO cells with deletion of the dihydrofolate reductase gene (dhfr) (CHO/DG44 cell) [Proceedings of the National Academy of Sciences, 77, 4216 (1980)], Lec13 cells that achieve lectin resistance [Somatic Cell and Molecular genetics, 12, 55 (1986)] , CHO cells with deletion of α1,6-fucosyltransferase gene (International Publication No. 2005/035586, International Publication No. 2002/31140), rat YB2/3HL.P2.G11.16Ag.20 cells (ATCC No.: CRL1662) and so on.

In addition, proteins such as enzymes involved in the synthesis of the intracellular nucleotide sugar GDP-fucose, and N-acetylene in which the 1-position of fucose is bound to the reducing end of the N-glycosidic complex sugar chain by an α bond can also be used. Proteins such as enzymes that modify the sugar chain at the 6-position of glucosamine, or host cells with reduced or absent activities such as proteins involved in transporting intracellular nucleotide sugar GDP-fucose to the Golgi apparatus, such as α1,6-fucos Glycosyltransferase gene-deleted CHO cells (International Publication No. 2005/035586, International Publication No. 2002/31140) and the like.

After introduction of the expression vector, by culturing in an animal cell culture medium containing reagents such as G418 sulfate (hereinafter referred to as G418), a transformant that stably expresses the gene recombinant antibody is selected (Japanese Patent Laid-Open No. 2-257891 ). ).

As the medium for animal cell culture, RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium (manufactured by JRH), EX-CELL302 medium (manufactured by JRH), EX-CELL325 medium ( JRH Corp.), IMDM medium (Invitrogen Corp.), Hybridoma SFM medium (Invitrogen Corp.), or a medium to which various additives such as FBS are added. The obtained transformed strain is cultured in a medium, whereby the accumulated gene recombinant antibody is expressed in the culture supernatant. The expression level and antigen-binding activity of the genetically recombinant antibody in the culture supernatant can be measured by ELISA or the like. In addition, the expression level of the recombinant antibody produced by the transformed strain can be enhanced by using the DHFR amplification system (Japanese Patent Laid-Open No. 2-257891 ).

The recombinant antibody can be purified from the culture supernatant of the transformed strain using a protein A column [Principles and Operations of Monoclonal Antibodies, 3rd Edition, Academic Press (1996), Antibodies: A Laboratory Manual, Cold Spring Hong Kong Laboratory (1988)]. Moreover, it is also possible to combine the methods used for protein purification, such as gel filtration, ion exchange chromatography, and ultrafiltration, for purification.

Polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting method [Principle and Operation of Monoclonal Antibodies, 3rd Edition, Academic Press (1996), Antibodies: Laboratory Manual, Cold Spring Harbor Laboratory (1988)] etc. to determine the molecular weight of the H chain, L chain or the whole antibody molecule of the purified recombinant antibody.

3. Design of Bispecific Antibodies 3-1. The N-terminal bispecific antibody of the present invention can be produced by designing an IgG portion comprising a second antigen-binding domain and a first antigen-binding domain, respectively, and linking these to design a bispecific antibody .

3-1(1). Design of IgG fraction The IgG part can be obtained by the following methods: obtaining the monoclonal antibody using the method described in 1. above, determining the cDNA sequences of the CDRs and variable regions of each antibody using the method described in 2. above, and designing the CDRs containing the antibody or can be obtained. The IgG portion of the variable region.

3-1(2). Design of the first antigen-binding domain When the first antigen-binding domain includes the CDR or variable region of the antibody, it can be prepared by the following method: determine the DNA sequence of the CDR or variable region of the antibody using the methods described in 1. and 2. above, and design the DNA sequence including the first antigen binding domains. As such a first antigen-binding domain, it is also possible to use a single-chain one in which VH and VL are directly bound, such as scFv or the like, or a single-chain one in which VH and VL are bound through a suitable linker, such as Fab and dsFv, etc., which are expressed as double-chains and designed as S-S bonds after expression. Combined form, or VHH, etc. The antigen-binding activity of the first antigen-binding domain can be evaluated by the above-described method, and those that maintain the antigen-binding activity can be selected.

3-2. The C-terminal bispecific antibody of the present invention can be produced by designing an IgG portion comprising a first binding domain and a second antigen binding domain by the same method as in 3-1. Design bispecific antibodies by ligation and so on.

4. Preparation of bispecific antibodies 4-1. Preparation of N-terminal bispecific antibody Specifically, the N-terminal bispecific antibody in which the first antigen-binding domain and the second antigen-binding domain are Fab and share four light chains can be produced as follows.

It can be prepared by synthesizing DNA encoding a polypeptide formed by linking VH-CH1 of the first antigen-binding domain and VH comprising the IgG portion of the second antigen-binding domain, synthesizing DNA encoding the VL of each antibody, The DNA is integrated into the vector for expression of the gene recombinant antibody described in the above 2.(2) to express the bispecific antibody. When the first antigen-binding domain and the IgG moiety are linked via a linker, the synthesis of DNA encoding the linker sequence is also included.

4-2. Production of N-terminal bispecific antibody whose first antigen-binding domain is other than Fab (1) The bispecific antibody whose first antigen-binding domain is VHH can be specifically prepared as follows. It can be produced by synthesizing DNA encoding a polypeptide formed by linking VHH and VH of the IgG part, synthesizing DNA encoding the VL of the IgG part, and integrating these DNAs into the genetic recombinant antibody described in 2.(2) above The bispecific antibody is expressed in a vector for expression. Where the VHH and IgG moieties are linked via a linker, the synthesis of DNA encoding the linker sequence is also included.

(2) A bispecific antibody in which the first antigen-binding domain is a polypeptide other than the above (1) and (2) containing scFv, dsFv, or CDRs can be specifically prepared as follows. It can be produced by synthesizing a DNA obtained by linking the DNA encoding the first antigen-binding domain and the DNA encoding the VH of the IgG moiety when the first antigen-binding domain is single-stranded. When the first antigen-binding domain is an association comprising two single-chain polypeptides, a single-chain polypeptide constituting the first antigen-binding domain is synthesized by linking a single-chain polypeptide that encodes the VH of the IgG moiety, and the DNA encoding the VH portion is also synthesized. DNA of another single-chain polypeptide of the first antigen-binding domain. In addition, DNA encoding the VL of the IgG portion was also synthesized. When the first antigen-binding domain and the IgG moiety are linked via a linker, the synthesis of DNA encoding the linker sequence is also included. These DNAs are integrated into the vector for expression of the recombinant antibody described in 2.(2) above to express the bispecific antibody.

(3) Preparation of bispecific antibody whose first antigen-binding domain is other than the above When the first antigen-binding domain is a polypeptide other than the above, the bispecific antibody of the present invention can be specifically prepared as follows. It can be produced by synthesizing a DNA obtained by linking the DNA encoding the first antigen-binding domain and the DNA encoding the VH of the IgG moiety when the first antigen-binding domain is single-stranded. When the first antigen-binding domain is an aggregate comprising two or more single-chain polypeptides, one of the single-chain polypeptides constituting the first antigen-binding domain is synthesized by linking with the DNA encoding the VH of the IgG part, and DNA encoding the remaining single-chain polypeptides constituting the first antigen-binding domain was also synthesized. In addition, DNA encoding the VL of the IgG portion was also synthesized. In the case where the IgG moiety is linked to the first antigen-binding domain via a linker, the synthesis of DNA encoding the linker sequence is also included. These DNAs are integrated into the vector for expression of the recombinant antibody described in 2.(2) above to express the bispecific antibody.

In addition to the method of using the fusion tumor described in 1. above, the antigen-binding domain can also be isolated and obtained by techniques such as phage display method and yeast display method [Emmanuelle Laffy et al., "Human Antibodies", 14 , 33-55 (2005)].

Furthermore, in the case of producing a bispecific antibody comprising a plurality of VHs and a single VL (also referred to as a light chain-sharing bispecific antibody), in order to make each antigen-binding site contained in the bispecific antibody specific for each Then, screening by phage display method is performed to select each VH that is most suitable for a single VL.

Specifically, first, after immunizing an animal with the first antigen by the method described in 1. above, a fusion tumor is produced from the spleen, and the DNA sequence encoding the first antigen-binding site is cloned. Next, animals are immunized with a second antigen, and a cDNA gene pool is prepared from their spleen, from which DNA encoding the amino acid sequence of VH is obtained by PCR.

Next, a phage library expressing an scFv formed by linking the VH obtained by immunization with the second antigen and the VL of the first antigen-binding site was prepared, and by panning using the Phage of scFv that specifically binds the antigen. The DNA sequence encoding the amino acid sequence of the VH of the second antigen binding site is cloned from the selected phage.

Furthermore, when the first antigen-binding domain is Fab, an amino acid sequence encoding a polypeptide formed by linking the VH of the first antigen-binding site and the VH of the second antigen-binding site via CH1 or via CH1 and a linker is designed The DNA sequence and the DNA sequence encoding the amino acid sequence of a single VL are inserted into, for example, the vector for expression of recombinant antibodies described in 2.(2) above, whereby the bispecific antibody of the present invention can be constructed the performance carrier.

4-3. Preparation of C-terminal Bispecific Antibody The C-terminal side bispecific antibody can be produced by producing an antibody expression vector into which a DNA sequence encoding an appropriately designed polypeptide is inserted by the same method as in 4-1. and 4-2. above, and making it express the polypeptide.

5. Activity evaluation of the bispecific antibody of the present invention or its antibody fragment Activity assessment of purified bispecific antibodies or fragments of the bispecific antibodies can be performed as follows.

The binding activity of the bispecific antibody of the present invention to a cell line expressing at least one of TfR and GPC3 can be measured using the binding assay system described in 1.(7) above.

CDC activity or ADCC activity against cells expressing at least one of TfR and GPC3 can be measured by well-known assay methods [Cancer Immunology, Immunotherapy, 36, 373 (1993)].

The anti-cellular activity (also referred to as proliferation inhibitory activity) of the bispecific antibodies of the present invention can be determined as follows. For example, cells are seeded in a 96-well plate, and antibodies are added. After a period of incubation, the cells are reacted with WST-8 reagent (manufactured by Dojindo Co., Ltd.), and the absorbance at 450 nm is measured using a plate reader to measure the cell viability. . In addition, other WST method (Total Superoxide Dismutase Assay Kit with WST) or MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method, [ ³ H] thymidine incorporation method to determine cell viability.

Signal transmission from TfR or GPC3 into cells can be assessed by detecting phosphorylation of intracellular proteins by Western blotting or the like.

6. Treatment of diseases using the bispecific antibody of the present invention or the bispecific antibody fragment The bispecific antibody or the bispecific antibody fragment of the present invention can be used for the treatment of diseases related to at least one of TfR and GPC3, preferably diseases in which expressing cells of TfR and GPC3 are involved. Examples of diseases related to at least one of TfR and GPC3 include malignant tumors and cancer.

Examples of malignant tumors and cancers include colorectal cancer, colorectal cancer, lung cancer, breast cancer, glioma, malignant melanoma, thyroid cancer, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, Gastric cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, esophageal cancer, liver cancer, head and neck cancer, squamous cell cancer, skin cancer, urinary tract cancer, bladder cancer, prostate cancer, choriocarcinoma, pharyngeal cancer Carcinoma, laryngeal cancer, pleuroma, androblastoma, endometrial hyperplasia, endometriosis, embryonal histoma, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retina blastoma, astrocytoma, neurofibroma, oligodendritic cell tumor, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteosarcoma, leiomyosarcoma and Wilms tumor.

Therapeutic drugs containing the bispecific antibody of the present invention or the bispecific antibody fragment or the derivative thereof may only contain the antibody or the antibody fragment or the derivative thereof as an active ingredient, and are usually combined with pharmacological methods. One or more kinds of carriers that are scientifically acceptable are mixed together and provided in the form of a pharmaceutical preparation produced by a method known in the field of formulation technology.

As an administration route, oral administration, or parenteral administration, such as intraoral, intratracheal, intrarectal, subcutaneous, intramuscular, intravenous, etc., are mentioned, for example. Examples of the administration form include sprays, capsules, lozenges, powders, granules, syrups, emulsions, suppositories, injections, ointments, and patches. Commonly used excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, dissolution aids, preservatives, colorants, flavoring agents can be used and stabilizers, etc., various formulations are produced by conventional methods.

Examples of excipients include lactose, fructose, glucose, corn starch, sorbitol, crystalline cellulose, sterilized water, ethanol, glycerol, physiological saline, and buffers. As a disintegrating agent, starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, synthetic magnesium silicate, etc. are mentioned, for example.

As a binding agent, methyl cellulose or its salt, ethyl cellulose, acacia, gelatin, hydroxypropyl cellulose, polyvinyl pyrrolidone, etc. are mentioned, for example. As a lubricant, talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil, etc. are mentioned, for example.

Examples of stabilizers include amino acids such as arginine, histidine, lysine, and methionine, human serum albumin, gelatin, dextran 40, methylcellulose, sodium sulfite, Sodium sulfite etc.

Examples of other additives include syrup, petrolatum, glycerin, ethanol, propylene glycol, citric acid, sodium chloride, sodium nitrite, and sodium phosphate.

Examples of formulations suitable for oral administration include emulsions, syrups, capsules, troches, powders, granules, and the like.

Liquid preparations such as emulsions or syrups are prepared using water, saccharides such as sucrose, sorbitol, or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, and parabens. Preservatives such as esters, and flavors such as strawberry flavor and peppermint are produced as additives.

Capsules, lozenges, powders or granules are prepared using excipients such as lactose, glucose, sucrose or mannitol, disintegrating agents such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, Binders such as hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, or plasticizers such as glycerin are produced as additives.

Examples of formulations suitable for parenteral administration include injections, suppositories, sprays, and the like. Injectables are prepared using a carrier or the like containing saline solution, glucose solution, or a mixture of the two.

Suppositories are produced using carriers such as cocoa butter, hydrogenated fats, or carboxylic acids. The spray is produced using a carrier that does not irritate the recipient's oral cavity and tracheal mucosa, and disperses the monoclonal antibody or its antibody fragment of the present invention into fine particles for easy absorption. As a carrier, lactose, glycerol, etc. are mentioned, for example. Moreover, it can also manufacture in the form of aerosol or dry powder. Furthermore, the exemplified components as additives in formulations suitable for oral administration may also be added to the above-mentioned parenteral formulations.

The effective amount when administered in the form of a combination of the effective amount of the bispecific antibody of the present invention, a suitable diluent and a pharmacologically acceptable carrier is 0.0001 mg to 100 mg per 1 kg body weight on average, with an interval of 2 Administer once every day to 8 weeks.

7. A method for diagnosing diseases using the bispecific antibody of the present invention or the bispecific antibody fragment Use the bispecific antibody of the present invention or the bispecific antibody fragment to detect or measure cells expressing at least one of TfR and GPC3, whereby diseases associated with at least one of TfR and GPC3 can be diagnosed, preferably TfR and GPC3 Diseases in which cells are involved.

For example, the diagnosis of a malignant tumor or cancer, which is a disease related to at least one of TfR and GPC3, can be performed by detecting or measuring at least one of TfR and GPC3 as follows.

First, using the bispecific antibody of the present invention or the bispecific antibody fragment or derivatives thereof, the following immunological methods are used to conduct at least TfR and GPC3 assays on biological samples collected from a plurality of healthy and normal organisms The detection or determination of one is to investigate the presence of at least one of TfR and GPC3 in biological samples of healthy normal subjects.

Next, the presence of at least one of TfR and GPC3 in the biological samples of the test subjects was similarly investigated, and the presence of the presence was compared with that of healthy normal subjects. A test subject is diagnosed with cancer when the presence of at least one of TfR and GPC3 in the test subject is increased compared to a healthy normal subject. The diagnosis of other diseases related to at least one of TfR and GPC3 can also be diagnosed by the same method.

The so-called immunological method refers to a method for detecting or measuring the amount of antibody or antigen by using labeled antigen or antibody. For example, a radioactive substance-labeled immunoantibody method, an enzyme immunoassay method, a fluorescence immunoassay method, a luminescence immunoassay method, a Western blot method, or a physicochemical method can be mentioned.

Examples of the radioactive substance-labeled immunoantibody method include, for example, a method in which an antigen or a cell expressing the antigen is reacted with the bispecific antibody or the antibody fragment of the present invention, and then a radiolabeled anti-immunoglobulin antibody or antibody is bound to the antibody. After the fragments are reacted, measurement is carried out using a scintillation counter or the like.

As an enzyme immunoassay method, for example, a method of reacting an antigen or a cell expressing the antigen with the bispecific antibody of the present invention or the bispecific antibody fragment, and further reacting with a labeled anti-immunoglobulin antibody or After reacting with the bound fragments, the color-developing pigment is measured by an absorptiophotometer. For example, a sandwich ELISA method etc. are mentioned.

As the label used in the enzyme immunoassay, a known enzyme label can be used ["Enzyme Immunoassay", Academy of Medicine (1987)]. For example, an alkaline phosphatase label, a peroxidase label, a luciferase label, or a biotin label is used.

The sandwich ELISA method is a method in which an antigen to be detected or measured is captured after binding an antibody to a solid phase, and the captured antigen is reacted with a second antibody. In this ELISA method, two kinds of antibodies or antibody fragments are prepared for binding to the antigen to be detected or measured, and these antigen-binding sites are different, wherein the first antibody or antibody fragment is previously adsorbed on a well plate (eg, 96 Å). well plate), and then label the second antibody or antibody fragment with fluorescent substances such as FITC, enzymes such as peroxidase, or biotin. In the well plate adsorbed with the above-mentioned antibodies, the cells isolated from the living body or their fragmented liquid, tissue or their fragmented liquid, cell culture supernatant, serum, pleural effusion, ascites or eye fluid are reacted, and then the labeled The antibody or antibody fragment reacts, and the corresponding detection reaction is carried out according to the labeling substance. Antigen concentrations in test samples were calculated based on calibration curves prepared from serial dilutions of antigens of known concentrations.

As the antibody used in the sandwich ELISA method, either a polyclonal antibody or a monoclonal antibody can be used, and an antibody fragment such as Fab, Fab', or F(ab) ₂ can also be used. The combination of the two antibodies used in the sandwich ELISA method can be a combination of monoclonal antibodies or antibody fragments that bind to different epitopes, or a combination of polyclonal antibodies and monoclonal antibodies or antibody fragments thereof.

As a fluorescent immunoassay method, for example, it is described in literature ["Principle and Operation of Monoclonal Antibodies", 3rd Edition, Academic Press (1996), "Handbook of Monoclonal Antibodies", Kodansha Science and Technology (1987)], etc. method of determination. As the label used in the fluorescent immunoassay, a known fluorescent label can be used ["Fluorescent Antibody Method", Soft Science Press (1983)]. For example, FITC or RITC, etc. are used.

As a luminescence immunoassay, for example, it can be measured by the method described in the literature ["Bioluminescence and Chemiluminescence Clinical Examination 42", Hirokawa Shoten (1998)] and the like. As the label used in the luminescence immunoassay, a well-known luminophore label, for example, acridinium ester or rophene, can be used.

As a Western blot method, it was determined by using SDS (sodium dodecyl sulfate)-PAGE (Polyacrylamide Gel Electrophoresis) ["Antibody: A Laboratory Manual", Cold Spring Harbor Laboratory (1988)] After dividing the antigen or antigen-expressing cells, etc., the gel is blotted to a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose membrane, and the membrane is bound to the antigen-binding antibody or antibody. Fragments are reacted with anti-IgG antibodies or antibody fragments labeled with fluorescent substances such as FITC, enzymes such as peroxidase, or biotin, and the labels are visualized. An example is shown below.

First, cells or tissues expressing polypeptides containing the desired amino acid sequence are lysed, and the amount of protein in each band is set to 0.1-30 μg under reducing conditions, and electrophoresis is performed by SDS-PAGE. Next, the protein after electrophoresis was transferred to a PVDF membrane and reacted with PBS containing 1-10% BSA (hereinafter referred to as BSA-PBS) at room temperature for 30 minutes to perform blocking operation. Here, react with the bispecific antibody of the present invention, wash with PBS containing 0.05-0.1% Tween-20 (Tween-PBS), and react with peroxidase-labeled goat anti-mouse IgG for 2 hours at room temperature . The antigen is detected by washing with Tween-PBS, and detecting the antibody-bound band using ECL Western blot detection reagent (manufactured by Amersham, Inc.) or the like. As the antibody used for detection by the Western blotting method, an antibody capable of binding to a polypeptide that does not retain a native stereostructure was used.

As a physicochemical method, for example, by binding at least one of TfR and GPC3 as antigens to the bispecific antibody of the present invention or the bispecific antibody fragment to form an aggregate, the aggregate is detected. As other physical and chemical methods, capillary method, one-dimensional immunodiffusion method, immunoturbidimetric method or latex immunoturbidimetric method can also be used ["Summary of Clinical Examination", Jinyuan Publishing (1998)] and so on.

In the latex immunoturbidimetric method, a carrier with a particle size of about 0.1-1 μm is used, such as an antibody or an antigen-sensitized polystyrene latex. If the antigen-antibody reaction is triggered by the corresponding antigen or antibody, the scattered light in the reaction solution will increases, the transmitted light decreases. The change is detected in the form of absorbance or integrating sphere turbidity, thereby determining the antigen concentration, etc. in the test sample.

On the other hand, the detection or measurement of cells expressing at least one of TfR and GPC3 can be performed by known immunological detection methods, preferably by immunoprecipitation method, immunocytostaining method, immunohistochemical staining method or fluorescent antibody staining method Wait.

As an immunoprecipitation method, after reacting cells expressing at least one of TfR and GPC3 with the bispecific antibody of the present invention or its antibody fragment, protein G Sepharose or the like is added with an immunoglobulin-specific binding ability. A carrier that precipitates the antigen-antibody complex.

Or it can be carried out by the following method. First, the bispecific antibody of the present invention or the bispecific antibody fragment of the present invention was immobilized on a 96-well plate for ELISA, and then blocked with BSA-PBS. Next, BSA-PBS is removed, washed sufficiently with PBS, and then reacted with a lysate of cells or tissues expressing at least one of TfR and GPC3. The immunoprecipitate was extracted from the well-washed well plate with the sample buffer for SDS-PAGE, and detected by the above-mentioned Western blotting.

The so-called immune cell staining method or immunohistochemical staining method refers to the following method: after treating cells or tissues expressing antigens with surfactants or methanol as appropriate to improve the penetration of antibodies, react with the bispecific antibodies of the present invention, Furthermore, after reacting with an anti-immunoglobulin antibody or a binding fragment thereof that has been subjected to a fluorescent label such as FITC, an enzyme label such as peroxidase, or a biotin label, the label is visualized and observed under a microscope. In addition, fluorescent antibody staining can be performed by reacting fluorescently labeled antibodies with cells and analyzing them by flow cytometry [Principle and Operation of Monoclonal Antibodies, 3rd Edition, Academic Press (1996), "Monoclonal Antibody Laboratory Manual", Kodansha Science and Technology (1987)] to implement the detection. In particular, the bispecific antibody of the present invention or the bispecific antibody fragment can detect at least one of TfR and GPC3 expressed on the cell membrane by fluorescent antibody staining.

In addition, in the fluorescent antibody staining method, when using the FMAT8100HTS system (manufactured by Applied Biosystems), etc., the formed antibody-antigen complex and the free antibody or antigen that does not participate in the formation of the antibody-antigen complex can be omitted. The amount of antigen or antibody is determined by separation. [Example]

Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to the following Example.

[Example 1] Preparation of soluble human and monkey TfR antigens, and soluble human and monkey GPC3 antigens 1. Preparation of soluble antigens of human TfR and monkey TfR The extracellular domain proteins of human and monkey TfR (hereinafter referred to as FLAG-Fc-human TfR and FLAG-Fc-monkey TfR, respectively) with FLAG-Fc added to the N-terminus were prepared by the method described below and added to the N-terminus. The protein in the extracellular domain of human TfR with a His tag (hereinafter referred to as His-human TfR).

The nucleotide sequence encoding the extracellular region of human TfR is represented by SEQ ID NO: 1, the amino acid sequence deduced from the nucleotide sequence is represented by SEQ ID NO: 2, the nucleotide sequence encoding the extracellular region of monkey TfR is represented by SEQ ID NO: 3, and SEQ ID NO: 4 shows the amino acid sequence estimated from the base sequence.

(1) Production of expression vectors for FLAG-Fc-human TfR and FLAG-Fc-monkey TfR Based on the human TfR gene sequence (Genbank accession number: NM_003234.2, SEQ ID NO: 5; the amino acid sequence encoded by the gene is represented by SEQ ID NO: 6), the extracellular region of human TfR comprising the nucleotide sequence represented by SEQ ID NO: 1 was synthesized the gene fragment.

Using the Infusion-HD colonization kit (manufactured by Clontech), the nucleotide sequence shown in SEQ ID NO: 1 was inserted into an INPEP4 vector (manufactured by Biogen-IDEC) containing a FLAG tag and the Fc region of human IgG to prepare FLAG-Fc- Human TfR expression vector.

By the same method, based on the gene sequence of monkey TfR (SEQ ID NO: 7; the amino acid sequence encoded by the gene is represented by SEQ ID NO: 8), an extracellular region containing monkey TfR comprising the nucleotide sequence represented by SEQ ID NO: 3 was prepared The FLAG-Fc-monkey TfR expression vector of the gene fragment.

(2) Preparation of FLAG-Fc-human TfR and FLAG-Fc-monkey TfR proteins Using the FreeStyle (trademark) 293 expression system (manufactured by Thermo Fisher), the FLAG-Fc-human TfR expression vector produced in (1) was introduced into HEK293 cells and cultured, and the protein was expressed in the transient expression system. Five days after the introduction of the vector, the culture supernatant was collected and filtered through a membrane filter with a pore size of 0.22 μm (manufactured by Millipore Corporation).

This culture supernatant was used for affinity purification using protein A resin (MabSelect, manufactured by GE Healthcare). Use Dulbecco's phosphate buffered saline [D-PBS(-) without calcium and magnesium, liquid; hereinafter referred to as D-PBS(-). Nacalai Tesque Co., Ltd.] The protein adsorbed by protein A was washed, eluted with 100 mM sodium citrate buffer (pH 3.5), and recovered into a tube containing 2M Tris-HCl (pH 8.5).

Next, the solvent was replaced with D-PBS(-) by ultrafiltration using VIVASPIN (manufactured by Sartorius), followed by filtration sterilization using Millex-Gv (manufactured by Millipore) with a pore size of 0.22 μm to prepare FLAG-Fc - Human TfR protein.

By the same method, FLAG-Fc-monkey TfR protein was produced using the FLAG-Fc-monkey TfR expression vector produced in (1).

The absorbance at a wavelength of 280 nm was measured, and the concentration of the obtained protein was calculated using the absorbance coefficient estimated from the amino acid sequence of each protein.

(3) Production of His-human TfR expression vector The base sequence shown in SEQ ID NO: 1 was inserted into a suitable restriction enzyme site of the pCI-OtCMV vector containing the His tag, thereby obtaining the expression vector of His-human TfR.

(4) Production of His-human TfR protein Using the Expi293 (trademark) expression system (manufactured by Thermo Fisher), the His-human TfR expression vector produced in (3) was introduced into Expi293F cells and cultured, and the protein was expressed in the transient expression system. Four days after the introduction of the vector, the culture supernatant was collected and filtered through a membrane filter with a pore size of 0.22 μm (manufactured by Millipore Corporation).

This culture supernatant was used for affinity purification using nickel resin (cOmplete His tag purification resin, manufactured by Sigma-Aldrich). The nickel-adsorbed protein was washed with a 20 mM sodium phosphate buffer containing 5 mM imidazole included in the His buffer set (manufactured by GE Healthcare), and then eluted with 250 mM imidazole.

Next, the eluate was replaced with D-PBS(-) by ultrafiltration using VIVASPIN (manufactured by Sartorius), followed by filtration sterilization using a membrane filter Millex-Gv (manufactured by Millipore) with a pore size of 0.22 μm to prepare His- Human TfR protein. The absorbance at a wavelength of 280 nm was measured, and the concentration of the obtained protein was calculated using the absorbance coefficient estimated from the amino acid sequence of each protein.

2. Preparation of soluble antigens of human GPC3 and monkey GPC3 (1) Production of expression vectors for human GPC3-mFc and mouse GPC3-mFc Expression vectors of human GPC3-mFc and mouse GPC3-mFc in which the Fc region of mouse IgG was bound to the C-terminus of human and mouse GPC3 proteins were prepared by the method described below.

The amino acid sequence of the full length of human GPC3 was obtained from the base sequence of the human GPC3 gene (Genbank accession number: NM_004484), and converted into codons most suitable for expression in mammalian cells to obtain the base sequence encoding the full length of human GPC3. Using the nucleotide sequence encoding the full length of GPC3 as a template, a DNA fragment of soluble human GPC3 was obtained by polymerase chain reaction (PCR). Furthermore, PCR was performed using a vector encoding mouse IgG as a template to obtain a DNA fragment of mouse Fc (hereinafter also referred to as mFc). Using the Infusion-HD colonization kit (manufactured by Clontech), a base sequence (SEQ ID NO: 9) linked to the C-terminus of soluble human GPC3 containing a signal sequence with mFc was inserted into pCI vector (manufactured by Promega). , and the expression vector of human GPC3-mFc was obtained. SEQ ID NO: 10 shows the amino acid sequence without the signal sequence among the amino acid sequences deduced from the base sequence of human GPC3-mFc.

By the same method, using the nucleotide sequence of the mouse GPC3 gene (Genbank accession number: NM_016697), a mouse GPC3-mFc into which the nucleotide sequence of the mouse GPC3-mFc (SEQ ID NO: 11) containing the signal sequence was inserted was prepared the performance carrier. SEQ ID NO: 12 shows the amino acid sequence without the signal sequence among the amino acid sequences deduced from the base sequence of mouse GPC3-mFc.

(2) Preparation of human GPC3-mFc and mouse GPC3-mFc Using the expression vector produced in (1), human GPC3-mFc and mouse GPC3-mFc were produced by the same method as in 1. (2).

Furthermore, 4 days after the introduction of the vector, the culture supernatant was recovered, and the protein adsorbed by protein A was washed with D-PBS (-), and then washed with 20 mM sodium citrate and 50 mM sodium chloride buffer (pH value). 3.4) Dissolution was carried out and recovered into a tube containing 1M sodium phosphate buffer (pH 7.0).

(3) Production of expression vectors for human GPC3-rFc and mouse GPC3-rFc Expression vectors of human GPC3-rFc and mouse GPC3-rFc in which the Fc region of rabbit IgG was bound to the C-terminus of human and mouse GPC3 proteins were prepared by the method described below.

By the same method as (1), a DNA fragment of soluble human GPC3 was obtained. Furthermore, PCR was performed using a vector encoding rabbit IgG as a template to obtain a DNA fragment of rabbit Fc (hereinafter also referred to as rFc). Using the Infusion-HD colonization kit (manufactured by Clontech), a base sequence (SEQ ID NO: 13) linked to the C-terminus of soluble human GPC3 containing a signal sequence with rFc was inserted into pCI vector (manufactured by Promega). , and the expression vector of human GPC3-rFc was obtained. SEQ ID NO: 14 shows the amino acid sequence without the signal sequence among the amino acid sequences deduced from the base sequence of human GPC3-rFc.

By the same method, the expression vector of mouse GPC3-rFc into which the nucleotide sequence (SEQ ID NO: 15) containing the signal sequence of mouse GPC3-rFc was inserted was produced. SEQ ID NO: 16 shows the amino acid sequence without the signal sequence among the amino acid sequences deduced from the base sequence of mouse GPC3-rFc.

(4) Preparation of human GPC3-rFc and mouse GPC3-rFc Using the expression vector produced in (3), human GPC3-rFc and mouse GPC3-rFc were produced by the same method as in (2).

(5) Production of human GPC3-GST vector An expression vector of human GPC3-GST in which GST was added to the C-terminus of human GPC3 protein was prepared by the method described below.

The GPI anchor additional sequence and the signal sequence were removed from the nucleotide sequence of the human GPC3 gene (Genbank accession number: NM_001164618), and the amino acid sequence of soluble human GPC3 represented by SEQ ID NO: 17 was obtained. The GST amino acid sequence was added to the C-terminus of the soluble human GPC3 amino acid sequence described in SEQ ID NO: 17 to prepare the human GPC3-GST amino acid sequence described in SEQ ID NO: 18. Based on the amino acid sequence of human GPC3-GST, the codons most suitable for expression in mammalian cells were converted to obtain the nucleotide sequence of human GPC3-GST described in SEQ ID NO: 19. The full-length nucleotide sequence of human GPC3-GST was synthesized and inserted into a suitable site of a pCI vector (manufactured by Promega) containing a signal sequence to produce a human GPC3-GST expression vector.

(6) Production of human GPC3-GST Using the expression vector of human GPC3-GST produced in (5), human GPC3-GST was produced by the same method as in (2).

In addition, glutathione resin (Glutathione Sepharose 4B, manufactured by GE Healthcare) was used for affinity purification of the culture supernatant. The glutathione-adsorbed protein was washed with D-PBS(-), and then eluted with 50 mM Tris-HCl and 10 mM reduced glutathione (pH 8.0).

(7) Production of the expression vector of the modified version of the heparan sulfate additional site of human GPC3 Based on the amino acid sequence of soluble human GPC3 described in SEQ ID NO: 17, the serine residue at position 495 and the serine residue at position 509, which are known as additional sites for heparan sulfate, were replaced by Alanine residue, thereby obtaining the amino acid sequence of soluble human GPC3 (hereinafter referred to as human GPC3-AA) without addition of heparin sulfate. Based on the amino acid sequence of human GPC3-AA, the codons most suitable for expression in mammalian cells were converted to obtain the base sequence of human GPC3-AA.

The full-length nucleotide sequence of human GPC3-AA was synthesized and inserted into a suitable restriction enzyme site of a pCI vector (manufactured by Promega) containing a signal sequence, a Flag tag and human Fc (hereinafter referred to as hFc), thereby producing human GPC3- The expression vector of AA-Flag-hFc (SEQ ID NO: 20 indicates the base sequence including the signal sequence). SEQ ID NO: 21 shows the amino acid sequence without the signal sequence among the amino acid sequences of human GPC3-AA-Flag-hFc estimated from the base sequence.

(8) Preparation of Heparan Sulfate Additional Site Modification of Human GPC3 Using the expression vector prepared in (7), by the same method as in (2), a heparan sulfate-added site-modified version of human GPC3, human GPC3-AA-Flag-hFc, was prepared.

(9) Preparation of biotinylated human GPC3-hFc and biotinylated monkey GPC3-hFc Biotinylated human GPC3-hFc and monkey GPC3-hFc (manufactured by ACRO Biosystems) were biotinylated using EZ-Link Sulfo-HNS-LC-Biotin, No-Weight Format (manufactured by Thermo Fisher Scientific) to obtain biotinylated human GPC3 -hFc and biotinylated monkey GPC3-hFc.

[Example 2] Preparation of human or monkey TfR membrane expressing CHO cells By the method described below, human TfR membrane-expressing CHO cells or monkey TfR membrane-expressing CHO cells (hereinafter referred to as human TfR/CHO cells or monkey TfR/CHO cells, respectively) were produced.

The human TfR gene represented by SEQ ID NO: 5 was cloned in pKANTEX (described in the specification of US Pat. No. 6,423,511) to obtain a human TfR expression vector for membrane expression pKANTEX-full-length human TfR.

The obtained expression vector pKANTEX-human TfR full-length was introduced into CHO cells and cultured to express the protein in a transient expression system. After gene introduction, single-cell colonization was performed to obtain human TfR/CHO cells.

By the same method, the monkey TfR gene represented by SEQ ID NO: 7 was cloned to obtain monkey TfR/CHO cells.

Cells stained with carboxyluciferin succinimidyl ester (CFSE) were prepared by the following method. Human TfR/CHO cells and monkey TfR/CHO cells were recovered by stripping with 0.02% EDTA-PBS (manufactured by Nacalai Tesque), then CFSE (manufactured by Sigma-Aldrich) was added to a final concentration of 0.2 μM, and the reaction was carried out at room temperature for 10 minutes. , D-PBS(-) containing 10% fetal bovine serum (manufactured by Nacalai Tesque) was added, and the cells were incubated at 37°C for 10 minutes. The cells after the reaction were centrifuged and washed with 1% BSA-PBS (manufactured by Nacalai Tesque) containing 0.02% EDTA and 0.05% NaN ₃ . The obtained CFSE-stained cells were cryopreserved in CELLBANKER1 (manufactured by Sanya Kogyo Co., Ltd.).

[Example 3] Acquisition of anti-TfR antibody 1. Preparation of the TfR immunized human antibody M13 phage library with the base sequence encoding the light chain as the A27 sequence In order to obtain monoclonal antibodies against human TfR, human antibody-producing animals were immunized with FLAG-Fc-human TfR mixed with adjuvant for 4 to 5 immunizations. Serum was recovered from the immunized animal, and the binding activity against human TfR was determined by ELISA to confirm the increase in antibody titer.

RNA was extracted from spleen cells or peripheral blood mononuclear cells (PBMC) obtained from immunized animals using RNeasy Mini kit (manufactured by QIAGEN), and cDNA was amplified by SMARTer RACE A kit (manufactured by Clontech) was used to amplify cDNA, and further a VH gene fragment was amplified by PCR. This VH gene fragment and the A27 sequence containing the nucleotide sequence represented by SEQ ID NO: 22, which is a human antibody germ-line sequence, were inserted into phagemid pCANTAB 5E (manufactured by Amersham Pharmacia), and Escherichia coli TG1 (Lucigen, Inc.) Production) transformation to obtain the coliform gene bank.

Furthermore, the A27 sequence encodes the light chain variable region (VL) of a human antibody comprising the amino acid sequence represented by SEQ ID NO: 23, and CDR1, 2 and 3 of the VL are represented by SEQ ID NO: 24, 25 and 26 (respectively, respectively). Denote the amino acid sequence of LCDR1, 2 and 3).

Amplify the obtained coliform gene library and infect it with VCSM13 Interference Resistant Helper Phage (manufactured by Agilent Technologies), thereby obtaining a VL gene containing the A27 sequence and a VH gene for gene libraryization The TfR immunized human antibody M13 phage library.

2. Obtaining an anti-TfR monoclonal antibody whose base sequence encoding the light chain is the A27 sequence Using the TfR immunized human antibody M13 phage library obtained in 1., an anti-TfR monoclonal antibody containing the VL encoded by the A27 sequence was obtained by the following phage display method.

(1) Protein panning using a protein immobilization tube Human TfR (manufactured by BBI Solutions Co., Ltd.) was immobilized, and the unbound site of human TfR was blocked with SuperBlock blocking buffer (manufactured by Thermo Fisher Co., Ltd.), and the MAXISORP STARTUBE (manufactured by NUNC Co., Ltd.) subjected to the above operations was combined with human antibody M13 phage library. React at room temperature for 1 to 2 hours. Then, after washing three times with D-PBS(-) and PBS containing 0.1% Tween20 (hereinafter referred to as PBS-T. manufactured by Wako Pure Chemical Industries, Ltd.), 0.1 M Gly-HCl (pH 2.2) was used for washing three times each. ) to dissolve the phage, and the eluate was neutralized with 2M Tris-HCl (pH 8.5).

(2) Cell panning using human TfR/CHO cells or monkey TfR/CHO cells The CHO cells and the human antibody M13 phage pool were reacted on ice for 30 minutes, the cells were removed by centrifugation, and the supernatant was recovered to prepare a phage solution after reacting with the CHO cells. After the phage solution was reacted with the CFSE-stained human TfR/CHO cells obtained in Example 2 on ice for 30 minutes to 1 hour, the supernatant was removed by centrifugation.

The cells were washed with D-PBS(-) (hereinafter referred to as SM buffer) containing 5% fetal bovine serum, 1 mM EDTA and 0.1% NaN ₃ , and an anti-M13 antibody (GE Healthcare) was added as a primary antibody. Company), react on ice for 30 minutes. The supernatant was removed by centrifugation, and the cells were washed several times with SM buffer. APC-labeled goat anti-mouse IgG (H+L) antibody (manufactured by Southern Biotech) was added as a secondary antibody, and the cells were placed on ice. React for 30 minutes.

The supernatant was removed by centrifugation, and the cells were washed several times with SM buffer. 7-Aminoactinomycin D (AAD) (manufactured by BD Biosciences) was added, and after reacting on ice for 10 minutes, 7-AAD-negative living cells were sorted by flow cytometry (manufactured by BD Bioscience, FACS AriaIII). And CFSE positive components.

When an APC-positive cell fraction was detected as a cell fraction obtained by concentrating phage binding to human TfR/CHO cells, the cell fraction was further sorted. Phage was eluted from the selected cells with 0.1 M Gly-HCl (pH 2.2), and the obtained eluate was neutralized with 2 M Tris-HCl (pH 8.5). The eluted phage was infected with TG1 competent cells and expanded.

Next, cell panning using monkey TfR/CHO cells was performed by the same method.

(3) Acquisition of anti-human TfR antibody The above-mentioned operation (1) or (2) was repeated two or three times to concentrate phages showing antibody molecules that specifically bind to human and monkey TfR. The concentrated phages were infected with TG1 and inoculated on SOBAG well plates (2.0% trypsin, 0.5% yeast extract, 0.05% NaCl, 2.0% glucose, 10 mM MgCl ₂ , 100 μg/mL ampicillin and 1.5% agar) to make colony formation.

The colonies are planted, and after culture, they are infected with VCSM13 anti-interference helper phage, and cultured again to obtain a single phage.

Using the obtained monoclonal phage solution, clones that bind to both human and monkey TfR were selected by the following flow cytometric analysis.

The monoclonal phage solution was added to the human or monkey TfR/CHO cells prepared in Example 2, and after reacting at 4° C. for 30 minutes, the supernatant was removed by centrifugation. After washing the cells with SM buffer, anti-M13 antibody (manufactured by GE Healthcare) was added as a primary antibody, and the cells were reacted on ice for 30 minutes.

The supernatant was removed by centrifugation, the cells were washed several times with SM buffer, and PE/Cy5.5-labeled goat F(ab') ₂ anti-mouse IgG H&L antibody (manufactured by Abcam) was added as a secondary antibody. ) and reacted on ice for 30 minutes. The supernatant was removed by centrifugation, the cells were washed several times with SM buffer, and then the fluorescence intensity of each cell was measured by a flow cytometer (Millipore, Guava Easy Cyte HT).

Sequence analysis was performed on the phage-presented antibody clones which were shown to bind to both human TfR/CHO cells and monkey TfR/CHO cells according to the obtained results. The amino acid sequence deduced from the entire nucleotide sequence encoding VH of each obtained anti-TfR antibody, and the amino acid sequence of CDR1-3 (hereinafter sometimes also referred to as HCDR1-3) of VH are shown in SEQ ID NOS: in Table 1.

[Table 1] Sequence numbering of anti-TfR antibody VH Breeding name amino acid sequence VH HCDR1 HCDR2 HCDR3 TfR1071 Serial number 27 Serial number 28 Serial number 29 Serial number 30 TfR1007 Serial number 31 Serial number 32 Serial number 33 Serial number 34

3. Acquisition of TfR1071 modified antibody The phage display method was used to obtain an amino acid sequence comprising the amino acid residue at position 1 of the VH amino acid sequence of TfR1071 described in SEQ ID NO: 27, which was replaced by a tyrosine residue with a glutamic acid residue ( An anti-TfR antibody in which the amino acid sequences of CDRs 1 to 3 of the VH of SEQ ID NO: 35) were modified (hereinafter, the antibody in which the amino acid sequence of the VH of TfR1071 was modified was referred to as a TfR1071 modified antibody). Table 2 shows the amino acid sequence deduced from the base sequence encoding VH of the obtained antibody, and the amino acid sequences of CDRs 1 to 3.

[Table 2] Breeding name amino acid sequence VH CDR1 CDR2 CDR3 TfR1071_201 Serial number 36 Serial number 37 Serial number 38 Serial number 39 TfR1071_202 Serial number 40 Serial number 41 Serial number 42 Serial number 43 TfR1071_203 Serial number 44 Serial number 45 Serial number 46 Serial number 47 TfR1071_204 Serial number 48 Serial number 49 Serial number 50 Serial number 51 TfR1071_301 Serial number 52 Serial number 53 Serial number 54 Serial number 55 TfR1071_302 Serial number 56 Serial number 57 Serial number 58 Serial number 59 TfR1071_303 Serial number 60 Serial number 61 Serial number 62 Serial number 63 TfR1071_304 Serial number 64 Serial number 65 Serial number 66 Serial number 67 TfR1071_305 Serial number 68 Serial number 69 Serial number 70 Serial number 71 TfR1071_306 Serial number 72 Serial number 73 Serial number 74 Serial number 75 TfR1071_307 Serial number 268 Serial number 269 Serial number 270 Serial number 271

4. Construction of a vector expressing anti-TfR antibody (1) Construction of the obtained anti-TfR antibody expression vector A soluble IgG expression vector incorporating the gene of the anti-TfR antibody obtained in 2.(3) was prepared by the method described below.

First, the A27 sequence encoding the common VL was sub-colonized to N5KG4PE (IDEC Pharmaceuticals) or N5KG4PE R409K (described in International Publication No. 2006/033386).

Thereafter, the VH gene of the anti-TfR antibody shown in Table 1 was sub-cultured to N5KG4PE or N5KG4PE R409K, and the Ser residue at position 228 in the EU index of the heavy chain constant region of human IgG4PE (human IgG4) was obtained and replaced. The constant region of the human IgG4 remodeled antibody in which the Leu residue at position 235 was replaced by Glu) or the Ser residue at position 228 in the EU index of human IgG4 PE R409K (the heavy chain constant region of human IgG4) Expression vector of anti-TfR monoclonal antibody with the constant region of Pro, the Leu residue at position 235 by Glu, and the Arg residue at position 409 by Lys.

(2) Construction of expression vector of anti-TfR monoclonal antibody TfR435 The expression vector was prepared by using the anti-TfR monoclonal antibody TfR435 described in International Publication No. 2012/153707 as a TfR neutralizing antibody as a positive control antibody for the anti-TfR antibody. SEQ ID NO: 76 shows the amino acid sequence of VH of TfR435, and SEQ ID NO: 77 shows the amino acid sequence of VL.

The genes encoding the VH and VL of TfR435 were synthesized and subcultured into the N5KG1 vector (described in International Publication No. 2003/033538) to obtain the expression vector of the anti-TfR monoclonal antibody TfR435 containing the constant region of human IgG1.

(3) Construction of expression vector of TfR1071 modified antibody (a) Preparation of expression vector for TfR1071 amino acid-modified antibody shown in Table 2 The base sequence encoding VH of the TfR1071 remodeled antibody shown in Table 2 and the base sequence encoding the A27 sequence of VL as the shared light chain were sub-selected into the pCI-OtCAG-G4PE R409K vector (pCI manufactured by Promega Corporation). The nucleotide sequence encoding the constant region of human IgG4PE R409K and the restriction enzyme site required for expression of human antibody gene are inserted into the vector) to make the expression vector of TfR amino acid modified antibody. The expression and preparation of the TfR1071 amino acid modified antibody was carried out according to the method described in Example 7.

(b) Production of expression vectors for TfR1071 modified antibodies P98D, P98E and P98K Expression vectors of the TfR1071-modified antibodies P98D, P98E and P98K shown in Table 3 were prepared by the same method as in (a) above.

The so-called P98D refers to the substitution of the amino acid residue at the 1st position in the amino acid sequence of the VH of TfR1071 represented by SEQ ID NO: 27 from a tyrosine residue to a glutamic acid residue, and the EU index as the 98th A modified antibody in which P (proline) of the amino acid residue at position 1 is replaced by D (aspartic acid). P98E and P98K are remodeled antibodies also defined in the same way.

[Table 3] Amino acid sequences of VH and HCDR1-3 of TfR1071 modified antibodies P98D, P98E and P98K The name of the modified antibody VH HCDR1 HCDR2 HCDR3 P98D Serial number 78 Serial number 79 Serial number 80 Serial number 81 P98E Serial number 82 Serial number 83 Serial number 84 Serial number 85 P98K Serial number 86 Serial number 87 Serial number 88 Serial number 89

[Example 4] Acquisition of anti-GPC3 antibody 1. Preparation of human naive antibody M13 phage library The VH gene fragment was amplified by PCR from cDNA derived from human PBMC. The VH gene fragment and the VL gene fragment containing the nucleotide sequence of the A27 sequence represented by SEQ ID NO: 22, which is a human antibody germline sequence, were inserted into the phagemid pCANTAB 5E (manufactured by Amersham Pharmacia) and the tag sequence was changed to FLAG- The plasmid obtained by transforming Escherichia coli TG1 (manufactured by Lucigen) into the vector obtained with the His tag and the trypsin recognition sequence was obtained.

The obtained plasmid was infected with VCSM13 anti-interference helper phage (manufactured by Agilent Technologies) to obtain a human primary antibody M13 phage library containing the VL gene including the nucleotide sequence of the A27 sequence and the gene library of the VH gene.

2. Preparation of GPC3 immunized human antibody M13 phage library with the base sequence encoding the light chain as A27. Human antibody-producing mice [Ishida & Lonberg, 2000 IBC's 11th Antibody Engineering, Abstract 2000] ; Ishida, I. et al., "Cloning & Stem Cells", 4, 91-102 (2002) and Ishida Gong (2002) Experimental Medicine 20, 6, 846-851] with 20 μg/ Human GPC3-GST, human GPC3-mFc, mouse GPC3-mFc, human GPC3-rFc, or mouse GPC3-rFc, or human GPC3- Fc (manufactured by ACRO Biosystems) or mouse GPC3-Fc (manufactured by ACRO Biosystems) was administered four times in total. Aluminium hydroxide (Alum) gel (0.25 mg/only or 2 mg/only) and inactivated pertussis bacteria suspension (manufactured by Nacalai Tesque) (1×10 ⁹ /only) were added as adjuvant only during the primary immunization. ).

The second immunization was performed 2 weeks after the initial immunization, the third immunization was performed 1 week later, the final immunization was performed 2 weeks after the third immunization, and the autopsy was performed 4 days after the final immunization, and surgical removal was performed. Take lymph nodes or spleen. After the extracted lymph node or spleen was homogenized, the cells were transferred into a tube through a cell strainer (manufactured by Falcon), and centrifuged to pellet the cells. The obtained spleen cells were mixed with erythrocyte removal reagent (manufactured by Sigma-Aldrich), reacted in a hot water bath at 37°C for 1 minute, diluted with DMEM medium (manufactured by Sigma-Aldrich), and further centrifuged.

Using the enhanced RNeasy Mini Plus kit (manufactured by QIAGEN), RNA was extracted from the obtained lymph node cells or spleen cells, and cDNA was amplified using the SMARTer RACE cDNA Amplification Kit (manufactured by Clontech). The VH gene fragment was amplified by PCR. The VH gene fragment and the VL gene fragment containing the nucleotide sequence of the A27 sequence represented by SEQ ID NO: 22, which is a human antibody germline sequence, were inserted into the phagemid pCANTAB 5E (manufactured by Amersham Pharmacia) and the tag sequence was changed to FLAG- The vector obtained by the His tag and the trypsin recognition sequence was transformed into Escherichia coli TG1 (manufactured by Lucigen) to obtain the Escherichia coli gene library.

The obtained coliform gene library was amplified to infect the VCSM13 anti-interference helper phage (manufactured by Agilent Technologies), thereby obtaining the GPC3 immunized human antibody M13 containing the VL gene including the A27 sequence and the VH gene to realize the gene library. Phage library.

3. Obtaining the anti-GPC3 monoclonal antibody whose base sequence encoding the light chain is the A27 sequence Using 1. the obtained human primary antibody M13 phage library or 2. the obtained GPC3 immunized human antibody M13 phage library, the following phage display method was used to obtain an anti-GPC3 monoclonal containing VL containing the amino acid sequence encoded by the A27 sequence. strain antibody.

(1) Obtaining the culture supernatant of a single strain of Escherichia coli Human GPC3-AA-FLAG-hFc and biotinylated human GPC3-Fc prepared in 2.(8) and (9) of Example 1 were immobilized on MAXISORP STARTUBE (manufactured by NUNC) by the method described below.

First, human GPC3-AA-FLAG-hFc was immobilized on MAXISORP STARTUBE and blocked with SuperBlock blocking buffer. Next, streptavidin (manufactured by Thermo Fisher) was immobilized on the MAXISORP STARTUBE (manufactured by NUNC), blocked with SuperBlock blocking buffer (manufactured by Thermo Fisher), and then bound to biotinylated human GPC3-Fc. .

Human GPC3-AA-FLAG-hFc immobilized on MAXISORP STARTUBE and biotinylated human GPC3-Fc were reacted with human primary antibody M13 phage library or GPC3 immunized human antibody M13 phage library at room temperature for 1 to 2 hours, using D- After washing with PBS(-) and PBS containing 0.1% Tween20 (hereinafter referred to as PBS-T, manufactured by Wako Pure Chemical Industries, Ltd.), the phage was eluted with 0.25% trypsin (manufactured by Nacalai Tesque). The eluted phage was infected with TG1 competent cells and expanded.

After the obtained phage was reacted again with biotinylated human GPC3-Fc and human GPC3-AA-FLAG-hFc immobilized on MAXISORP STARTUBE, the tube was washed and the phage was eluted. This procedure is repeated to concentrate phages presenting antibody molecules that specifically bind to human GPC3 full-length or human GPC3-AA.

Plasmids were prepared from transformed E. coli obtained by infecting the concentrated phage with TG1. Using the prepared plasmid, the Mix&Go Competent Cells-TG1 strain (Mix&Go Competent Cells-Strain TG1) (manufactured by Zymo Research) was transformed, and inoculated on SOBAG well plates (2.0% trypsin, 0.5% yeast extract, 0.05% NaCl, 2.0% glucose, 10 mM MgCl ₂ , 100 μg/mL ampicillin and 1.5% agar) to form colonies.

The colony was inoculated, and after several hours of culture, 1 mM IPTG (manufactured by Nacalai Tesque) was added, and the culture was performed again to obtain a single-strain coliform culture supernatant.

(2) Selection of clones by ELISA method Using the single-strain E. coli culture supernatant obtained in (1), a colony that binds to both human GPC3 and monkey GPC3 was selected by the ELISA method described below.

Streptavidin (manufactured by Thermo Fisher) was immobilized on a MAXISORP well plate (manufactured by NUNC), blocked with 1% BSA-PBS (manufactured by Nacalai Tesque), and then combined with 2.(9 of Example 1). ) biotinylated human GPC3-Fc or biotinylated monkey GPC3-Fc. The culture supernatant was added to each well of the well plate, and after 60 minutes of reaction at room temperature, each well was washed three times with PBS-T.

Next, Penta His HRP conjugate (manufactured by QIAGEN) was diluted 1000-fold with 1% BSA-PBS, 50 μL was added to each well, and incubated at room temperature for 30 minutes. After washing the microplate three times with PBS-T, 50 μL of TMB color development matrix solution (manufactured by DAKO) was added to each well, and incubated at room temperature for 10 minutes. A 2N HCl solution (50 μL/well) was added to each well to stop the color reaction, and the absorbance at a wavelength of 450 nm (reference wavelength 570 nm) was measured using a plate reader (EnSpire, manufactured by PerkinElmer).

(3) Sequence analysis of the selected clones Sequence analysis was performed on the clones selected in (2) that bind to both human GPC3 and monkey GPC3, and the obtained results are shown in Table 4.

The clone name of the obtained GPC3 antibody, the amino acid sequence estimated from the entire nucleotide sequence encoding VH, and the sequence of the amino acid sequence of CDR1-3 (hereinafter also referred to as HCDR1-3) of VH The numbers are shown in Table 4.

[Table 4] Breeding name amino acid sequence VH HCDR1 HCDR2 HCDR3 G1015 Serial number 90 Serial number 91 Serial number 92 Serial number 93 G2002 Serial number 94 Serial number 95 Serial number 96 Serial number 97 G1036 Serial number 98 Serial number 99 Serial number 100 Serial number 101 G2133 Serial number 102 Serial number 103 Serial number 104 Serial number 105 G2067 Serial number 106 Serial number 107 Serial number 108 Serial number 109 G2103 Serial number 110 Serial number 111 Serial number 112 Serial number 113 G1119 Serial number 114 Serial number 115 Serial number 116 Serial number 117 G3005 Serial number 118 Serial number 119 Serial number 120 Serial number 121 G1016 Serial number 122 Serial number 123 Serial number 124 Serial number 125 G1054 Serial number 126 Serial number 127 Serial number 128 Serial number 129 G2038 Serial number 130 Serial number 131 Serial number 132 Serial number 133 G1018 Serial number 134 Serial number 135 Serial number 136 Serial number 137 G2072 Serial number 138 Serial number 139 Serial number 140 Serial number 141 G4068 Serial number 142 Serial number 143 Serial number 144 Serial number 145 G4045 Serial number 146 Serial number 147 Serial number 148 Serial number 149 G1104 Serial number 150 Serial number 151 Serial number 152 Serial number 153 G3117 Serial number 154 Serial number 155 Serial number 156 Serial number 157 G3024 Serial number 158 Serial number 159 Serial number 160 Serial number 161 G3018 Serial number 162 Serial number 163 Serial number 164 Serial number 165 G3062 Serial number 166 Serial number 167 Serial number 168 Serial number 169 G3177 Serial number 170 Serial number 171 Serial number 172 Serial number 173 G2024 Serial number 174 Serial number 175 Serial number 176 Serial number 177 G1137 Serial number 178 Serial number 179 Serial number 180 Serial number 181 G2017 Serial number 182 Serial number 183 Serial number 184 Serial number 185 G1107 Serial number 186 Serial number 187 Serial number 188 Serial number 189 G4025 Serial number 190 Serial number 191 Serial number 192 Serial number 193 G4009 Serial number 194 Serial number 195 Serial number 196 Serial number 197 G3008 Serial number 198 Serial number 199 Serial number 200 Serial number 201 G1048 Serial number 202 Serial number 203 Serial number 204 Serial number 205 G3042 Serial number 206 Serial number 207 Serial number 208 Serial number 209 G3033 Serial number 210 Serial number 211 Serial number 212 Serial number 213 G4037 Serial number 214 Serial number 215 Serial number 216 Serial number 217 G1122 Serial number 218 Serial number 219 Serial number 220 Serial number 221 G2011 Serial number 222 Serial number 223 Serial number 224 Serial number 225 G2025 Serial number 226 Serial number 227 Serial number 228 Serial number 229 G4033 Serial number 230 Serial number 231 Serial number 232 Serial number 233

The amino acid sequences of the VHs of G3062 (SEQ ID NO: 166), G3177 (SEQ ID NO: 170), G2024 (SEQ ID NO: 174) and G2017 (SEQ ID NO: 182) in the anti-GPC3 antibodies shown in Table 4 are contained in the CDR or framework regions N-type sugar chain attached amino acid sequence (N-X-S/T; X represents an amino acid residue other than P). Remodeled antibodies G3062_NYA, G3177_NTA, G2024_NNA and G2017_NNA were designed with this sequence removed. The amino acid sequences of VH and the amino acid sequences of HCDRs 1 to 3 of these modified antibodies are shown in Table 5.

The so-called G3062_NYA refers to a modified antibody obtained by substituting the N-type sugar chain additional amino acid sequence (NXS/T; X represents an amino acid residue other than P) contained in the VH of G3062 with NYA. G3177_NTA, G2024_NNA and G2017_NNA are remodeled antibodies also defined in the same way. [table 5] Breeding name amino acid sequence VH HCDR1 HCDR2 HCDR3 G3062_NYA Serial number 234 Serial number 235 Serial number 236 Serial number 237 G3177_NTA Serial number 238 Serial number 239 Serial number 240 Serial number 241 G2024_NNA Serial number 242 Serial number 243 Serial number 244 Serial number 245 G2017_NNA Serial number 246 Serial number 247 Serial number 248 Serial number 249

4. Preparation of anti-GPC3 antibody HN3-Fc Antibody HN3-Fc containing the variable region of the anti-GPC3 monoclonal antibody HN3 described in International Publication No. 2012/145469 was produced by the method described below. The amino acid sequence of the VH of HN3 is represented by SEQ ID NO: 250.

The VH and hinge of HN3 described in SEQ ID NO: 250 and the full-length nucleotide sequence encoding the Fc of human IgG4PE R409K described in SEQ ID NO: 251 were synthesized and inserted into a suitable site of a pCI vector (manufactured by Promega) to obtain an anti-GPC3 clone. Expression vector for antibody HN3-Fc.

[Example 5] Construction of expression vector of bispecific antibody binding to TfR and GPC3 Using the anti-TfR monoclonal antibody and anti-GPC3 monoclonal antibody obtained in Example 3 and Example 4, VH1 was an anti-GPC3 antibody with the structure shown in (A) to (C) of Fig. 1 by the following method. The VH and VH2 of the anti-TfR antibody VH are bispecific antibodies that bind to TfR and GPC3 (hereinafter referred to as N-terminal GPC3-TfR bispecific antibody, C-terminal bispecific antibody and GPC3-GS-TfR, respectively). Bispecific antibodies. Also, these are also collectively referred to as GPC3-TfR bispecific antibodies). The structure of the bispecific antibody adopts the structure described in International Publication No. 2009/131239.

The heavy chain of the N-terminal GPC3-TfR bispecific antibody prepared by the following procedure contains the VH of the anti-GPC3 antibody and the CH1 of the human IgG4 (the base sequence is represented by SEQ ID NO: 252, SEQ ID NO: 1) in this order from the N-terminal side. 253 represents amino acid sequence), VH of anti-TfR antibody, and CH (CH1, hinge, CH2 and CH3) of human IgG4PE R409K described in International Publication No. 2006/033386 (SEQ ID NO: 255 represents amino acid sequence).

The heavy chain of the C-terminal GPC3-TfR bispecific antibody contains the VH of the anti-GPC3 antibody, the CH of the human IgG4PE R409K, the VH of the anti-TfR antibody, and the CH1 of the human IgG4 (represented by SEQ ID NO: 258) in this order from the N-terminal side. base sequence, amino acid sequence represented by SEQ ID NO: 253).

In addition, the bispecific antibodies produced by the following steps all contain a light chain including VL encoded by the A27 sequence.

The heavy chain of the GPC3-GS-TfR bispecific antibody contains, in order from the N-terminal side, the VH of the anti-GPC3 antibody, the GS sequence [GGGGS (SEQ ID NO: 256)], the VH of the anti-TfR antibody, and the CH (CH1, hinge, CH2 and CH3).

Table 6 shows the names and structures of the bispecific antibodies produced, the selection of anti-GPC3 antibodies (VH1) and the selection of anti-TfR antibodies (VH2) used to produce the bispecific antibodies.

[Table 6] hN3-GS-TfR1071 Structure of Bispecific Antibodies VH1 VH2 Nt-G1015-TfR1071 Figure 1(A) G1015 TfR1071 Nt-G2002-TfR1071 Figure 1(A) G2002 TfR1071 Nt-G1036-TfR1071 Figure 1(A) G1036 TfR1071 Nt-G2133-TfR1071 Figure 1(A) G2133 TfR1071 Nt-G2067-TfR1071 Figure 1(A) G2067 TfR1071 Nt-G2103-TfR1071 Figure 1(A) G2103 TfR1071 Nt-G1119-TfR1071 Figure 1(A) G1119 TfR1071 Nt-G3005-TfR1071 Figure 1(A) G3005 TfR1071 Nt-G1016-TfR1071 Figure 1(A) G1016 TfR1071 Nt-G1054-TfR1071 Figure 1(A) G1054 TfR1071 Nt-G2038-TfR1071 Figure 1(A) G2038 TfR1071 Nt-G1018-TfR1071 Figure 1(A) G1018 TfR1071 Nt-G2072-TfR1071 Figure 1(A) G2072 TfR1071 Nt-G4068-TfR1071 Figure 1(A) G4068 TfR1071 Nt-G4045-TfR1071 Figure 1(A) G4045 TfR1071 Nt-G1104-TfR1071 Figure 1(A) G1104 TfR1071 Nt-G3117-TfR1071 Figure 1(A) G3117 TfR1071 Nt-G3024-TfR1071 Figure 1(A) G3024 TfR1071 Nt-G3018-TfR1071 Figure 1(A) G3018 TfR1071 Nt-G3062_NYA-TfR1071 Figure 1(A) G3062_NYA TfR1071 Nt-G3177_NTA-TfR1071 Figure 1(A) G3177_NTA TfR1071 Nt-G2024_NNA-TfR1071 Figure 1(A) G2024_NNA TfR1071 Nt-G1137-TfR1071 Figure 1(A) G1137 TfR1071 Nt-G2017_NNA-TfR1071 Figure 1(A) G2017_NNA TfR1071 Nt-G1107-TfR1071 Figure 1(A) G1107 TfR1071 Nt-G4025-TfR1071 Figure 1(A) G4025 TfR1071 Nt-G4009-TfR1071 Figure 1(A) G4009 TfR1071 Nt-G3008-TfR1071 Figure 1(A) G3008 TfR1071 Nt-G1048-TfR1071 Figure 1(A) G1048 TfR1071 Nt-G3042-TfR1071 Figure 1(A) G3042 TfR1071 Nt-G3033-TfR1071 Figure 1(A) G3033 TfR1071 Nt-G4037-TfR1071 Figure 1(A) G4037 TfR1071 Nt-G1122-TfR1071 Figure 1(A) G1122 TfR1071 Nt-G2011-TfR1071 Figure 1(A) G2011 TfR1071 Nt-G2025-TfR1071 Figure 1(A) G2025 TfR1071 Nt-G4033-TfR1071 Figure 1(A) G4033 TfR1071 Ct-G1015-TfR1071 Figure 1(B) G1015 TfR1071 Ct-G4068-TfR1071 Figure 1(B) G4068 TfR1071 Ct-G1054-TfR1071 Figure 1(B) G1054 TfR1071 Ct-G1137-TfR1071 Figure 1(B) G1137 TfR1071 Ct-G3008-TfR1071 Figure 1(B) G3008 TfR1071 Ct-G4045-TfR1071 Figure 1(B) G4045 TfR1071 Ct-G1122-TfR1071 Figure 1(B) G1122 TfR1071 Ct-G1104-TfR1071 Figure 1(B) G1104 TfR1071 Ct-G1048-TfR1071 Figure 1(B) G1048 TfR1071 Ct-G2025-TfR1071 Figure 1(B) G2025 TfR1071 Ct-G3042-TfR1071 Figure 1(B) G3042 TfR1071 HN3-GS-TfR1071 Figure 1(C) HN3 TfR1071

Furthermore, the amino acid sequence of the VH2 of the N-terminal GPC3-TfR bispecific antibody and the C-terminal GPC3-TfR bispecific antibody shown in Table 6 used the VH of the anti-TfR antibody TfR1071 described in SEQ ID NO: 27. The amino acid sequence in which the amino acid residue at position 1 of the amino acid sequence was replaced by a tyrosine residue with a glutamic acid residue (SEQ ID NO: 35). In the VH including the amino acid sequence represented by SEQ ID NO: 35, the amino acid sequences of CDRs 1 to 3 are represented by SEQ ID NOs: 28 to 30, respectively.

1. Production of expression vector for heavy chain of N-terminal GPC3-TfR bispecific antibody The expression vector for the heavy chain of the N-terminal GPC3-TfR bispecific antibody shown in Table 6 was prepared by the method described below.

The nucleotide sequence fragment encoding the VH of the anti-GPC3 antibody described in Table 4 and Table 5 was inserted into the appropriate restriction enzyme site of the pCI vector (manufactured by Promega) to obtain the heavy chain expression vector. The above pCI vector contains The nucleotide sequence encoding CH1 of human IgG4 described in SEQ ID NO: 253 (SEQ ID NO: 252), and the amino acid residue at the first position encoding the amino acid sequence of VH of TfR1071 described in SEQ ID NO: 27 are composed of tyrosine residues. The nucleotide sequence (SEQ ID NO: 257) of the amino acid sequence (SEQ ID NO: 35) in which the glutamic acid residues are substituted by the group, and the amino acid sequence encoding the polypeptide comprising CH of the human IgG4PE R409K described in SEQ ID NO: 255, and The 3' end contains the base sequence of the stop codon.

2. Production of expression vector for heavy chain of C-terminal GPC3-TfR bispecific antibody Expression vectors for the heavy chains of the C-terminal GPC3-TfR bispecific antibodies shown in Table 6 were prepared by the method described below.

The nucleotide sequence fragment encoding the VH of the anti-GPC3 antibody described in Table 4 and Table 5 was inserted into the appropriate restriction enzyme site of the pCI vector (manufactured by Promega) to obtain the heavy chain expression vector. The above pCI vector contains The nucleotide sequence (SEQ ID NO: 254) encoding the amino acid sequence of the polypeptide comprising the CH of human IgG4PE R409K described in SEQ ID NO: 255, and the first amino acid sequence encoding the VH of the anti-TfR antibody TfR1071 described in SEQ ID NO: 27 A base sequence (SEQ ID NO: 257) of the amino acid sequence (SEQ ID NO: 35) in which the amino acid residue at the position was replaced by a tyrosine residue with a glutamic acid residue, and encoding human IgG4 as a C-terminal side polypeptide The C-terminal side sequence gene of CH1 (SEQ ID NO: 258).

3. Production of expression vector for GPC3-GS-TfR bispecific antibody Expression vectors of GPC3-GS-TfR bispecific antibodies shown in Table 6 were prepared by the method described below.

The A27 sequence encoding the VL shared by the anti-TfR antibody obtained in Example 3 and the anti-GPC3 antibody obtained in Example 4 was sub-selected into the pCI-OtCAG-G4PE R409K vector (insert encoding human IgG4PE into the pCI vector manufactured by Promega). The base sequence of the constant region of R409K and the vector obtained by expressing the restriction enzyme site required for the human antibody gene). This vector is hereinafter referred to as pCI-A27.

Next, a nucleotide sequence encoding a polypeptide comprising the VH of the anti-GPC3 antibody HN3 represented by SEQ ID NO: 250, the GS linker and the VH of the anti-TfR antibody TfR1071 represented by SEQ ID NO: 27 in order from the N-terminus was fully synthesized, The sequence was cloned into pCI-A27 to obtain the expression vector for the GPC3-GS-TfR bispecific antibody.

[Example 6] Preparation of monoclonal antibody binding to TfR and bispecific antibody binding to TfR and GPC3 Using the antibody expression vectors prepared in Examples 3 and 5, anti-TfR monoclonal antibodies and GPC3-TfR bispecific antibodies were prepared by the following methods.

1. Production of anti-TfR antibody and GPC3-GS-TfR bispecific antibody Using the Expi293 (trademark) expression system (manufactured by Thermo Fisher), the antibody expression vector prepared in Example 3 or Example 5 was introduced into Expi293F cells, and 12 to 16 hours later, a transfection enhancer was added, and Transient expression systems express antibodies.

4 to 7 days after gene introduction, the culture supernatant was recovered, and after filtration, protein A purification, ultrafiltration and filter sterilization were carried out in the same manner as in 1.(2) of Example 1 to prepare an antibody solution. The absorbance of the obtained antibody solution at a wavelength of 280 nm was measured, and the concentration of purified antibody was calculated using the absorbance coefficient estimated from the amino acid sequence of each antibody.

2. Preparation of N-terminal GPC3-TfR bispecific antibody and C-terminal GPC3-TfR bispecific antibody Using the Expi293 (trademark) expression system (manufactured by Thermo Fisher), the heavy chain expression vector of the bispecific antibody prepared in Example 5 and pCI-A27 as the light chain expression vector were co-genes introduced into Expi293F cells. 1 Prepare the antibody solution in the same way, and calculate the concentration of the antibody.

[Example 7] Evaluation of the binding of GPC3-TfR bispecific antibody to TfR and GPC3 by ELISA The binding properties of each bispecific antibody prepared in Example 6 to each of human GPC3, monkey GPC3, and human TfR were evaluated by the ELISA method described below.

The antigenic protein was diluted with D-PBS(-) at a concentration of 2 μg/mL, and 50 μL/well was dispensed into Ni-NTA HisSorb Plates (manufactured by QIAGEN), and left to stand at room temperature for 1 hour. After washing the wells three times with 200 μL/well of D-PBS(-), 1% (w/v) BSA-PBS(-) (pH 7.0, manufactured by Nacalai Tesque Co., Ltd.) was added to the wells at 50 μL/well. , hereinafter referred to as BSA-PBS) diluted to 10 μg/mL of each GPC3-TfR bispecific antibody. As the antigen protein, human GPC3-His (manufactured by ACRO Biosystems), monkey GPC3-His (manufactured by ACRO Biosystems), or His-human TfR prepared in Example 1 was used.

After standing at room temperature for 1 hour, the well plate was washed three times with 200 μL/well of PBS-T, and 50 μL/well of 1% (w/v) BSA-PBS(-) (pH value) was added. 7.0, manufactured by Nacalai Tesque, hereinafter referred to as BSA-PBS) as a secondary antibody, peroxidase-labeled affinity-purified goat anti-human IgG (Fcγ fragment specific) (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ Fragment specific) (manufactured by Jackson ImmunoReseach Co., Ltd.) The resulting solution was diluted 1,000 times and left to stand for 1 hour.

After that, the plate was washed three times with 200 μL/well of PBS-T, then washed twice with 200 μL/well of D-PBS(-), and an equal amount of mixed substrate solution (50 μL/well) was added. For liquid A and liquid B of substrate reagent pack (manufactured by R&D systems), let stand for 5 minutes. The reaction was stopped by adding 50 μL/well of Stop solution (manufactured by R&D Systems), and the absorbance at 450 nm and 570 nm was measured using an EPOCH2 microplate reader (manufactured by Biotek). The value obtained by subtracting the absorbance at 570 nm from the absorbance at 450 nm obtained was calculated, and the obtained results are shown in FIGS. 2A to 2F .

As a negative control, the following were used: using ["Clin Cancer Res", 2005, 11(8), 3126-3135] described in ["Clin Cancer Res", 2005, 11 (8), 3126-3135] containing encoding anti-2,4-dinitrophenol (DNP) antibody The base sequence carrier was a human IgG4 antibody (having the constant region of human IgG4PE R409K; hereinafter abbreviated as anti-DNP antibody) produced according to the method described in 1. of Example 6.

Figures 2A and 2B show the results of measuring the binding of each GPC3-TfR bispecific antibody to human TfR, Figures 2C and 2D show the results of measuring the binding of each GPC3-TfR bispecific antibody to human GPC3, Figure 2E And FIG. 2F shows the results of measuring the binding of each GPC3-TfR bispecific antibody to monkey GPC3.

According to FIGS. 2A to 2F , it was shown that all GPC3-TfR bispecific antibodies bound to human GPC3, monkey GPC3 and human TfR.

[Example 8] Evaluation of TfR and GPC3 expression of cancer cell lines by flow cytometry The expression levels of TfR and GPC3 in HepG2 cells and HLE cells were evaluated by fluorescence-activated cell sorting (FACS) according to the following procedure. Fluorescently labeled anti-human CD71 antibody (manufactured by BD Pharmingen) was used to evaluate the expression level of TfR, and the anti-GPC3 antibody HN3-Fc prepared in Example 4 was used to evaluate the expression level of GPC3.

HepG2 cells or HLE cells were suspended in D-PBS(-) staining buffer (SB) containing 0.1% NaN ₃ , 1% FBS, and 1×10 ⁵ cells/well were dispensed into 96-well round-bottom well plates ( manufactured by Falcon). After centrifugation (2000 rpm, 4°C, 2 minutes), the supernatant was removed, PE-labeled anti-human CD71 antibody or HN3-Fc was added to the well plate, and the cells were incubated at ice temperature for 30 minutes. The cells to which PE-labeled anti-human CD71 antibody was added were washed twice with SB, suspended in SB, and the fluorescence intensity of each cell was measured by a flow cytometer FACS CANTO II (manufactured by BD). The cells after the addition of HN3-Fc were washed twice with SB, then Alexa Fluor (registered trademark) 488 goat anti-human IgG (H+L) (manufactured by Molecular Probes) was added as a secondary antibody, and the cells were incubated at ice temperature for 30 minutes. min, the fluorescence intensity was measured by the same method. Anti-DNP antibody was used as a negative control.

(A) of FIG. 3 shows the expression level of TfR in HepG2 cells, and (B) shows the expression level of TfR in HLE cells. (C) of FIG. 3 shows the expression level of GPC3 in HepG2 cells, and (D) shows the expression level of GPC3 in HLE cells.

According to (A) and (B) of FIG. 3 , both HepG2 cells and HLE cells moderately expressed TfR. According to (C) and (D) of FIG. 3 , HepG2 cells highly express GPC3, while HLE cells do not express GPC3.

Taken together, it is shown that HepG2 cells express both TfR and GPC3 on cells, and HLE cells express TfR but not GPC3 on cells.

[Example 9] Evaluation of cell proliferation inhibitory activity of anti-TfR antibody The anti-TfR antibodies TfR435 and TfR1071 prepared in Example 6 were evaluated for their growth-inhibitory activity on cancer cell lines by the following method.

HLE cells or HepG2 cells were suspended in DMEM medium (manufactured by Nacalai Tesque) containing 10% FBS, 0.9×10 ³ cells/well were seeded in a flat-bottom 96-well plate (manufactured by Falcon), and the cells were incubated at 37° C. under 5.0% carbon dioxide. Pre-incubation for 24 hours. After incubation, the test antibody was added at a final concentration of 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 or 10 μg/ml. The time point was assumed to be 0 days after culturing, and then the cells were cultured at 37°C and 5.0% carbon dioxide for 6 days. After 0 days and 6 days of culture, CellTiter-Glo (registered trademark) luminescent cell viability assay reagent (Luminescent Cell Vialbility Assay) (manufactured by Promega) was added, and a microplate reader (EnVision 2104 Multilabel Reader, PerkinElmer) was used. manufacture) to measure the luminous intensity.

The value of the obtained luminescence intensity reflects the amount of ATP (Adenosine triphosphate, adenosine triphosphate) in the living cells in each well. For each condition, three independent wells were used for evaluation, and the average value of the luminescence intensity (hereinafter referred to as the average luminescence intensity) was calculated. Anti-DNP antibody was used as a negative control. As a control group, medium-only wells to which no antibody was added were prepared. Based on the calculated average luminescence intensity, the cell viability relative to the control group (hereinafter also abbreviated as cell viability) was calculated by Equation 1, and the obtained results are shown in FIG. 4 . (A) of FIG. 4 shows the results of the evaluation of the cell proliferation activity of each antibody on HLE cells, and (B) shows the results of the evaluation of the cell proliferation activity of each antibody on HepG2 cells.

Cell survival rate relative to the control group [survival rate (% relative to the control group)] = (the average luminescence intensity of each sample after 6 days of culture - the average luminescence intensity of the control group after 0 days of culture)/(6 days of culture The average luminous intensity of the control group after that - the average luminous intensity of the control group after culturing for 0 days)...(Formula 1)

As shown in (A) and (B) of FIG. 4 , TfR435, which is a TfR neutralizing antibody, decreased the cell viability of both HLE cells and HepG2 cells compared to the anti-DNP antibody, indicating cell proliferation inhibitory activity. On the other hand, TfR1071 was equivalent to the anti-DNP antibody in the cell viability of HLE cells and the cell viability of HepG2 cells, indicating that it did not have cell proliferation inhibitory activity.

Taken together, it was shown that TfR1017 is a TfR non-neutralizing antibody that has no cell proliferation inhibitory activity despite binding to TfR on cancer cells.

[Example 10] Evaluation of cell proliferation inhibitory activity of GPC3-TfR bispecific antibody (1) Evaluation of cell proliferation activity as an indicator of cell survival rate Using the cell survival rate as an index, the growth inhibitory activity of each GPC3-TfR bispecific antibody prepared in Example 6 on cancer cell lines was evaluated by the same method as in Example 9.

The results obtained when the antibody concentration was 10 μg/mL are shown in FIGS. 5A to 5F . 5A to 5C show the results of evaluating the cell proliferation activity of each antibody on HLE cells, and FIGS. 5D to 5F show the results of evaluating the cell proliferation activity of each antibody on HepG2 cells.

As shown in FIG. 5A to FIG. 5C , each GPC3-TfR bispecific antibody was equivalent to the anti-DNP antibody in the cell viability of HLE cells, and showed no growth inhibitory activity on HLE cells. On the other hand, as shown in FIGS. 5D to 5F , among the GPC3-TfR bispecific antibodies used in the experiment, bispecific antibodies other than Ct-G3042-TfR1071 (Nt-G3042-TfR1071, Nt-G1048-TfR1071, Nt-G1104-TfR1071, Nt-G4045-TfR1071, Nt-G4068-TfR1071, Nt-G2072-TfR1071, Nt-G1018-TfR1071, Nt-G2038-TfR1071, Nt-G1054-TfR1071, Nt-fR1071, Nt-G1016 G3005-TfR1071, Nt-G1119-TfR1071, Nt-G2103-TfR1071, Nt-G2067-TfR1071, Nt-G2133-TfR1071, Nt-G1036-TfR1071, Nt-G2002-TfR1071, Nt-G1073-TfR1040-T TfR1071, Nt-G2025-TfR1071, Nt-G2011-TfR1071, Nt-G1122-TfR1071, Nt-G4037-TfR1071, Nt-G3033-TfR1071, Nt-G3008-TfR1071, Nt-G4009-TfR1071, Nt-G4025-TfR1071 Nt-G1107-TfR1071, Nt-G2017NNA-TfR1071, Nt-G1137-TfR1071, Nt-G2024NNA-TfR1071, Nt-G3177NTA-TfR1071, Nt-G3062NYA-TfR1071, Nt-G3018-TfR-1071, Nt-G3024 G3117-TfR1071, Ct-G2025-TfR1071, Ct-G1048-TfR1071, Ct-G1104-TfR1071, Ct-G1122-TfR1071, Ct-G4045-TfR1071, Ct-G3008-TfR1071, C1t-G1110710-T TfR1071, Ct-G4068-TfR1071, Ct-G1015-TfR1071 and HN3-GS-TfR1071) decreased the survival rate of HepG2 cells, and showed proliferation inhibitory activity on HepG2 cells.

As described in Example 8, HLE cells express TfR but not GPC3 on cells, on the other hand, HepG2 cells express both TfR and GPC3 on cells.

Therefore, it is shown that the GPC3-TfR bispecific antibody of the present invention does not inhibit cell proliferation when only binding to TfR on cancer cells, but inhibits cancer cell proliferation only when binding to TfR and GPC3 on cancer cells.

To sum up, it is shown that the GPC3-TfR bispecific antibody of the present invention does not inhibit cell proliferation in normal liver and other sites without GPC3 positive cells, and specifically inhibits cell proliferation in tumor sites where GPC3 positive cells exist.

In addition, as shown in FIG. 4(B), the anti-TfR antibody TfR1071 did not inhibit the proliferation of HepG2 cells. On the other hand, as shown in FIGS. 5D to 5F , the GPC3-TfR bispecific antibodies of the present invention prepared using the antigen binding domain of TfR1071 all inhibited the proliferation of HepG2 cells.

Therefore, it was shown that TfR1071 has cell proliferation inhibitory activity only by making a bispecific antibody with GPC3 antibody.

(2) Calculation of _IC50 of each antibody Based on the cell viability at each antibody concentration obtained in (1), JMP15 (manufactured by SAS Institute Japan) was used to calculate the half-maximum ratio of each GPC3-TfR bispecific antibody to HepG2 cells Inhibitory concentration ( _IC50 ). The obtained results are shown in Tables 7A to 7C.

[Table 7A] Antibody name IC50(μg/mL) TfR435 0.074 HN3-GS-TfR1071 0.083 Nt-G3042-TfR1071 NC Nt-G1048-TfR1071 1.794 Nt-G1104-TfR1071 0.614 Nt-G4045-TfR1071 0.386 Nt-G4068-TfR1071 0.248 Nt-G2072-TfR1071 0.241 Nt-G1018-TfR1071 0.24 Nt-G2038-TfR1071 0.237 Nt-G1054-TfR1071 0.223 Nt-G1016-TfR1071 0.203 Nt-G3005-TfR1071 0.184 Nt-G1119-TfR1071 0.159 Nt-G2103-TfR1071 0.156 Nt-G2067-TfR1071 0.147 Nt-G2133-TfR1071 0.135 Nt-G1036-TfR1071 0.119 Nt-G2002-TfR1071 0.106 Nt-G1015-TfR1071 0.102

[Table 7B] Antibody name IC50(μg/mL) TfR435 0.05 HN3-GS-TfR1071 0.036 Nt-G4033-TfR1071 NC Nt-G2025-TfR1071 NA Nt-G2011-TfR1071 1.477 Nt-G1122-TfR1071 1.006 Nt-G4037-TfR1071 0.746 Nt-G3033-TfR1071 0.534 Nt-G3008-TfR1071 0.267 Nt-G4009-TfR1071 0.265 Nt-G4025-TfR1071 0.228 Nt-G1107-TfR1071 0.217 Nt-G2017NNA-TfR1071 0.177 Nt-G1137-TfR1071 0.162 Nt-G2024NNA-TfR1071 0.133 Nt-G3177NTA-TfR1071 0.076 Nt-G3062NYA-TfR1071 0.064 Nt-G3018-TfR1071 0.062 Nt-G3024-TfR1071 0.059 Nt-G3117-TfR1071 0.056

[Table 7C] Antibody name IC50(μg/mL) TfR435 0.117 CtG3042-TfR1071 NC CtG2025-TfR1071 NC CtG1048-TfR1071 NC CtG1104-TfR1071 1.862 CtG1122-TfR1071 1.32 CtG4045-TfR1071 0.952 CtG3008-TfR1071 0.312 CtG1137-TfR1071 0.281 CtG1054-TfR1071 0.274 CtG4068-TfR1071 0.258 CtG1015-TfR1071 0.219

NCs in Tables 7A to 7C represent cell viability above 50%, so _IC50 cannot be calculated. NA in Table 7B indicates that the calculated 95% confidence interval for _IC50 is outside the treatment concentration domain, simulating off-label use.

According to Table 7A to Table 7C, it was confirmed that Nt-G1104-TfR1071, Nt-G4045-TfR1071, Nt-G4068-TfR1071, Nt-G2072-TfR1071, Nt-G1018-TfR1071, Nt-G2038-TfR1071, Nt-G1054-TfR1071 Nt-G1016-TfR1071, Nt-G3005-TfR1071, Nt-G1119-TfR1071, Nt-G2103-TfR1071, Nt-G2067-TfR1071, Nt-G2133-TfR1071, Nt-G1036-TfR1071, Nt-TfR1071, Nt-G2002 G1015-TfR1071, Nt-G3008-TfR1071, Nt-G4009-TfR1071, Nt-G4025-TfR1071, Nt-G1107-TfR1071, Nt-G2017NNA-TfR1071, Nt-G1137-TfR1071, Nt-G4017-TfR1024NNA TfR1071, Nt-G3062NYA-TfR1071, Nt-G3018-TfR1071, Nt-G3024-TfR1071, Nt-G3117-TfR1071, Ct-G4045-TfR1071, Ct-G3008-TfR1071, Ct-G1137-5CfR10 The _IC50 of Ct-G4068-TfR1071, Ct-G1015-TfR1071 and HN3-GS-TfR1071 was less _than 10 times that of the TfR neutralizing antibody TfR435.

[Example 11] Evaluation of the influence of HN3-GS-TfR1071 on the expression of TfR Using the down-regulation of the expression number of TfR on HepG2 cells as an index, the effect of HN3-GS-TfR1071 as a GPC3-GS-TfR bispecific antibody prepared in Example 6 on the expression of TfR on the cancer cell surface was evaluated in the following manner.

HepG2 cells were seeded at 0.5×10 ⁵ cells/well in a flat-bottom 6-well plate (manufactured by Falcon) containing DMEM medium (manufactured by Nacalai Tesque) containing 10% FBS, and pre-cultured at 37°C under 5.0% carbon dioxide for 24 days. Hour. After the incubation, the test antibody or HN3-Fc was added so that the final concentration would be 10 μg/ml, and the cells were incubated at 37° C. and 5.0% carbon dioxide for 24 hours. After that, the cells were detached, suspended in D-PBS(-) staining buffer (SB) containing 0.1% NaN ₃ , 1% FBS, and dispensed at 1×10 ⁵ cells/well to a round-bottom 96-well plate (Falcon manufactured by the company). After centrifugation (2000 rpm, 4°C, 2 minutes), the supernatant was removed, Alexa Fluor 488-labeled anti-human CD71 antibody (manufactured by eBiosience) was added to the well plate at 5 μg/mL, and incubated at ice temperature for 30 days. minute. After washing twice with SB, the cells were suspended in SB, and the fluorescence intensity of the cells was measured by a flow cytometer.

Next, using QIFIKIT (registered trademark) (manufactured by FUJIFILM Wako Pure Chemical Co., Ltd.), a calibration curve between the average number of antigen molecules per cell and the fluorescence intensity was prepared. Using this calibration curve, the average number of TfR molecules per cell at the time of each antibody addition was calculated. Anti-DNP antibody was used as a negative control.

The obtained results are shown in FIG. 6 . As shown in Figure 6, the average number of TfR molecules per cell by anti-DNP antibody or HN3-Fc was equivalent to that of the control group (media only), whereas HN3-GS-TfR1071 reduced the average number of TfR molecules per cell.

According to the results of Examples 10 and 11, HN3-GS-TfR1071 reduced the number of TfR molecules on the surface of GPC3-positive cancer cells, thereby inhibiting cell proliferation.

[Example 12] Epitope identification of anti-TfR antibody 1071 1. Production of known anti-TfR antibodies According to the sequence information described in International Publication No. 2014/189973, the VH of anti-TfR antibodies 15G11v5, 7A4v15 and 16F6v4 that bind to the apical domain (Apical domain) of human TfR, and the anti-TfR antibody 7G7v1 that recognizes the protease-like domain will be encoded and VL base sequences were cloned into pCI-OtCAG-G4PE R409K vector (the base sequence encoding the constant region of human IgG4PE R409K and the restriction enzyme site required for expression of human antibody gene were inserted into pCI vector manufactured by Promega Corporation the obtained vector) to prepare a vector for antibody expression. Using this antibody expression carrier, an antibody was produced by the same method as in Example 6.

2. Production of human TfR extracellular domain protein and human and mouse chimeric TfR extracellular domain protein By the same method as in 1.(3) and (4) of Example 1, a His-tagged human TfR extracellular domain protein (hereinafter referred to as His-huTfR) or the apical domain of His-huTfR was prepared. Or the chimeric protein obtained by replacing the protease-like domain with the corresponding region of mouse TfR (the amino acid sequence shown in GenBank accession number NP_001344227 or the amino acid sequence shown in SEQ ID NO: 259) (respectively marked as containing mouse apical structure) huTfR with mouse Apical domain (SEQ ID NO: 260) or huTfR with mouse protease-like domain (SEQ ID NO: 261)).

Similarly, a part of amino acid residues in the apical domain of His-huTfR was substituted with the corresponding amino acid residues of mouse TfR to prepare a chimeric TfR protein. The name of the produced chimeric TfR protein, the amino acid residue substitution sites in the amino acid sequence of human TfR, and the sequence number of the amino acid sequence of the chimeric TfR protein are shown in Table 8.

[Table 8] The name of the chimeric TfR protein Amino acid residue substitution sites in the amino acid sequence of human TfR amino acid sequence A02 D352S, S355A, D356R, K358N Serial number 262 A03 D245E, K261E Serial number 263 A04 D245E, K261E, D356R Serial number 264 A05 P249S, L274F Serial number 265 A07 M365L, V366E, E369Q Serial number 266 A14 D204Q, K205S, E369Q Serial number 267

Regarding the amino acid residue substitution sites in the amino acid sequence of human TfR shown in Table 8, for example, D352S, S355A, D356R, K358N of A02 means the amino acid sequence No. 6 in the amino acid sequence of human TfR shown by SEQ ID NO: 6. The aspartic acid residue at position 352 was replaced by a serine residue, the serine residue at position 355 was replaced by an alanine residue, and the aspartic acid residue at position 356 was replaced by an arginine residue , and the lysine residue at position 358 was replaced by an asparagine residue.

3. Epitope analysis of various TfR antibodies With respect to the anti-TfR antibodies prepared in Examples 3 and 6, human TfR/CHO prepared in Example 2 and anti-TfR antibodies prepared in the same manner as in Example 2 were evaluated by the following fluorescence-activated cell sorting (FACS) method. Reactivity of murine TfR/CHO.

Human TfR/CHO cells were suspended in D-PBS(-) staining buffer (SB) containing 0.1% NaN ₃ , 1% FBS, and 1×10 ⁵ cells/well were dispensed into round-bottom 96-well plates (Falcon manufactured by the company). After centrifugation (2000 rpm, 4°C, 2 minutes), the supernatant was removed, anti-TfR antibody was used as the primary antibody, and Alexa Fluor (registered trademark) 488 goat anti-human IgG (H+) was added as the secondary antibody. L) (manufactured by Molecular Probes), incubated at ice temperature for 30 minutes. After washing twice with SB, the cells were suspended in SB, and the fluorescence intensity of each cell was measured by a flow cytometer FACS CANTO II (manufactured by BD). Anti-DNP antibody was used as a negative control. The binding of anti-TfR antibodies to mouse TfR/CHO cells was similarly evaluated.

As a result, both TfR1007 and TfR1071 bound to human TfR, but not to mouse TfR. In addition, it is known that 15G11v5, 7A4v15, 16F6v4 and 7G7v1 produced in 1. above do not bind to mouse TfR (International Publication No. 2014/189973).

Furthermore, the binding activity of the anti-TfR antibody to the various chimeric TfRs prepared in 2. above was evaluated by the following ELISA method, and the binding domain and recognition epitope of the anti-TfR antibody were identified.

1% (w/v) BSA-PBS(-) (pH 7.0) without KCl was added to a 96-well immunoculture plate (manufactured by Nunc) on which the antigen protein was immobilized, hereinafter referred to as blocking solution, manufactured by Nacalai Tesque ), after blocking at room temperature, anti-TfR antibody diluted with the same solution was added and reacted at room temperature for 1 hour.

After that, it was washed with KCl-free 0.05% (w/v)-tPBS (1x) (pH 7.2) (hereinafter referred to as washing solution, manufactured by Nacalai Tesque Co., Ltd.), and anti-human IgG ( Fc) goat IgG Fab'-HRP (manufactured by IBL) was reacted at room temperature for 1 hour, washed with a washing solution, and added with 1-Step (registered trademark) Ultra TMB-ELISA substrate solution (manufactured by Thermo). The reaction was carried out at room temperature, 5N HCl solution was added to stop the color development reaction, and the absorbance at a wavelength of 450 nm (reference wavelength 570 nm) was measured using a plate reader (EPOCH2: manufactured by BioTek).

The obtained results are shown in Table 9.

[Table 9] Breed ELISA binding binding domain ELISA binding to human-mouse chimeric TfR huTfR apical domain binding site huTfR containing mouse apical domain huTfR containing mouse protease-like domain A02 A03 A04 A05 A07 A14 TfR1007 - ++ top - ++ + ++ - ++ A02, A07 TfR1071 - ++ top - ++ + ++ - ++ A02, A07 15G11v5 - ++ top - - - ++ + ++ A02, A03, A04 7A4v15 - ++ top - ++ + ++ + ++ A02 16F6v4 - ++ top - ++ + ++ ++ ++ A02 7G7v1 ++ - protease-like N/A N/A N/A N/A N/A N/A N/A

N/A in Table 9 indicates that no data was obtained. In addition, the binding activity of each TfR antibody to each chimeric TfR (value obtained by subtracting the absorbance at a reference wavelength of 570 nm from the absorbance at a wavelength of 450 nm) was relative to the binding activity of each TfR antibody to His-huTfR (from a wavelength of 450 nm). When the absorbance at nm minus the absorbance at the reference wavelength of 570 nm) is 0.5 or more, it is recorded as ++, when it is more than 0.2 and less than 0.5, it is recorded as +, and when it is less than 0.2, it is recorded as -, ++ or + judgment For binding, - is judged as not binding.

According to Table 9, 15G11v5, 7A4v15 and 16F6v4 bound to His-huTfR and huTfR containing a mouse protease-like domain, but did not bind to huTfR containing a mouse apical domain. On the other hand, 7G7v1 bound to His-huTfR and huTfR containing a mouse apical domain, but did not bind to huTfR containing a mouse protease-like domain. Therefore, it was confirmed that 15G11v5, 7A4v15, and 16F6v4 bound to the apical domain, and 7G7v1 bound to the protease-like domain.

According to Table 9, TfR1071 and TfR1007 bound to His-huTfR and huTfR containing a mouse protease-like domain, but did not bind to huTfR containing a mouse apical domain. Therefore, it was shown that TfR1071 and TfR1007 bind to the apical domain.

In addition, among the antibodies that bind to the apical domain, it can be seen that TfR1007 and TfR1071 bind to the amino acid residue replacement sites of A02 and A07, 15G11v5 binds to the amino acid residue replacement sites of A02, A03 and A04, 7A4v15 and 16F6v4 Binds to the amino acid residue replacement site of A02.

Therefore, it was shown that TfR1071 binds to 7 amino acid residues of D352, S355, D356, K358, M365, V366 and E369 in the amino acid sequence of human TfR represented by SEQ ID NO: 6. The same can be said for TfR1007.

[Example 13] Epitope Analysis of Anti-GPC3 Antibody In order to analyze the epitope of the anti-GPC3 antibody obtained in Example 4, the Fab of the anti-GPC3 antibody was used to evaluate whether it competes with the known anti-GPC3 antibody GC33 for binding to human GPC3 by enzyme-linked immunosorbent assay (ELISA).

First, according to the sequence information described in International Publication No. 2006/006693, a vector expressing anti-GPC3 antibody GC33 was prepared by the same method as in Example 4, and GC33 was prepared by the same method as in Example 6 using this vector.

Next, 1% (w/v) BSA-PBS(-) (pH 7.0) without KCl was added to a 96-well immunoculture plate (manufactured by Nunc) on which human GPC3-AA-Fc was immobilized, hereinafter referred to as blocking solution , manufactured by Nacalai Tesque Company), blocking at room temperature. The E. coli culture supernatant and GC33 of the anti-GPC3 antibody (test antibody) prepared in Example 4 were diluted with the same solution, and added to the above-mentioned culture plate. (hereinafter referred to as PBS-T, manufactured by Wako Pure Chemical Industries, Ltd.) was washed three times.

Next, Penta His HRP conjugate (manufactured by QIAGEN) was diluted 1000-fold with a blocking solution, 50 μL per well was added, and the cells were incubated at room temperature for 30 minutes. After washing the microplate three times with PBS-T, 50 μL of TMB color development matrix solution (manufactured by DAKO) was added to each well, and incubated at room temperature for 10 minutes. A 2N HCl solution (50 μL/well) was added to each well to stop the color development reaction, and the absorbance at 450 nm and 570 nm was measured using a plate reader (EnSpire, manufactured by PerkinElmer). The value obtained by subtracting the absorbance at 570 nm from the obtained absorbance at 450 nm (hereinafter abbreviated as absorbance) is calculated, and the obtained result is shown in (A) of FIG. 7 .

In addition, the relative value (%) of the absorbance of each test antibody in the presence of GC33 relative to the absorbance of each test antibody in the absence of GC33 was calculated [hereinafter referred to as +GC33/-GC33(%)], and the obtained results were shown as In Figure 7 (B). When +GC33/-GC33(%) is less than 70%, it is judged that the test antibody competes with GC33 for binding to human GPC3, and when +GC33/-GC33(%) is more than 70%, it is judged that the test antibody does not bind to GC33 Competitive binding to human GPC3.

According to (B) of Figure 7, G1048, G1104, G4045, G4068, G2072, G2038, G1054, G1016, G3005, G1119, G2103, G2067, G2133, G1036, G2002, G1015, G3033, G4009, G1137, G201 The +GC33/-GC33 (%) of G2024, G3177, G3062, G3018, G3024 and G3117 was more than 70%, which did not compete with GC33 for binding to human GPC3.

Therefore, it shows that G1048, G1104, G4045, G4068, G2072, G2038, G1054, G1016, G3005, G1119, G2103, G2067, G2133, G1036, G2002, G1015, G3033, G4009, G4025, G23017, G1706 , G3018, G3024 and G3117 bind to human GPC3 at different epitopes from GC33.

Moreover, according to FIG. 7(B), the +GC33/-GC33 (%) of G1018, G2025, G3008 and G1107 was less than 70%, and competed with GC33 for binding to human GPC3. Therefore, it was shown that G1018, G2025, G3008 and G1107 bind to human GPC3 at an epitope similar to that of GC33.

[Example 14] Preparation of bispecific antibody binding to TfR and GPC3 According to the method described in Example 5, the C-terminal GPC3-TfR bispecific antibody shown in FIG. 8 and the N-terminal GPC3-TfR bispecific antibody shown in FIGS. 9A to 9G were produced.

The GPC3-TfR bispecific antibody shown in Figure 8 is named according to the method of Ct-[the clone name of the anti-GPC3 antibody]-[the clone name of the anti-TfR antibody], indicating that it contains two heavy chains and four light chains. Bispecific antibody, the two heavy chains contain the VH of the anti-GPC3 antibody, the CH containing the amino acid sequence represented by SEQ ID NO: 255, the VH of the anti-TfR antibody, and the VH of the anti-TfR antibody, and the anti-GPC3 antibody represented by SEQ ID NO: 253. As for CH of the amino acid sequence, the four light chains contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 in this order from the N-terminal side.

In addition, the N-terminal GPC3-TfR bispecific antibody shown in FIGS. 9A to 9G has the structure shown in FIG. 1(A), and is based on Nt-[the colony name of the anti-GPC3 antibody]-[the anti-TfR antibody Colony name]. Indicates a bispecific antibody comprising 2 heavy chains and 4 light chains, the two heavy chains sequentially contain the VH of the anti-GPC3 antibody colonized from the N-terminus, and the amino acid sequence represented by SEQ ID NO: 253. CH1, the VH of the anti-TfR antibody, and the CH comprising the amino acid sequence represented by SEQ ID NO: 255, the four light chains from the N-terminal side sequentially contain VL comprising the amino acid sequence represented by SEQ ID NO: 23, and a sequence comprising: CL of the amino acid sequence represented by No. 272.

Tables 1 to 3 describe the sequence numbers of the amino acid sequences of the VH of the anti-TfR antibodies, and Tables 4 and 5 describe the sequence numbers of the amino acid sequences of the VH of the anti-GPC3 antibodies. Among them, the amino acid sequence of SEQ ID NO: 35 was used as the amino acid sequence of VH of TfR1071.

[Example 15] Evaluation of the binding of GPC3-TfR bispecific antibody to TfR and GPC3 By the same method as in Example 7, the binding properties of the GPC3-TfR bispecific antibody prepared in Example 14 to TfR and GPC3 were confirmed.

[Example 16] Evaluation of cell proliferation inhibitory activity of GPC3-TfR bispecific antibody By the same method as in Example 10, the cell growth inhibitory activity of the GPC3-TfR bispecific antibody prepared in Example 14 was confirmed.

[Example 17] Preparation of GPC3 knockdown HepG2 cells In order to investigate the effect of the amount of GPC3 expression on cells on the activity of the GPC3-TfR bispecific antibody of the present invention, cells with down-regulated GPC3 expression were prepared. Using SMARTvector GPC3 hEF1a-TurboGFP Particles (manufactured by Dharmacon), shRNA against GPC3 gene was introduced into HepG2 cells, and puromycin was used for selection, thereby constructing two types of cells with down-regulated GPC3 expression, named HepG2#28, HepG2#P05. Next, the expression of GPC3 on each of HepG2, HepG2#28, and HepG2#P05 cells was investigated by the following method. Anti-human GPC3 antibody mouse IgG1κ (manufactured by Enzo Life Sciences) was used as the primary antibody, and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 647 (manufactured by Invitrogen) was used as the secondary antibody. cytometry was performed. Beads supplied with QIFIKIT (manufactured by DAKO) were used as quantitative beads. The results are shown in FIG. 10 .

As shown in FIG. 10 , the expression level of GPC3 was down-regulated in sequence according to HepG2>HepG2#P05>HepG2#28, and cells whose expression level was continuously down-regulated were obtained. The expression level of TfR was equivalent to that of HepG2 as the parent strain.

[Example 18] Evaluation of the cell proliferation inhibitory activity of GPC3-TfR bispecific antibody on GPC3 knockdown HepG2 cells Using the cell viability as an index, the following N-terminal GPC3-TfR bispecific antibodies (27 strains) and C-terminal GPC3-TfR bispecific antibodies ( 6 strains) on the growth inhibitory activity of the GPC3 knockdown HepG2 cells prepared in Example 17. Nt-G4025-TfR1071 Nt-G2038-TfR1071 Nt-G1036-TfR1071 Nt-G3062NNA-TfR1071 Nt-G2002-TfR1071 Nt-G1137-TfR1071 Nt-G1119-TfR1071 Nt-G1107-TfR1071 Nt-G2133-TfR1071 Nt-G2072-TfR1071 Nt-G3008-TfR1071 Nt-G1015-TfR1071 Nt-G3005-TfR1071 Nt-G1016-TfR1071 Nt-G1054-TfR1071 Nt-G1104-TfR1071 Nt-G4045-TfR1071 Nt-G4009-TfR1071 Nt-G2067-TfR1071 Nt-G4068-TfR1071 Nt-G3117-TfR1071 Nt-G2017NNA-TfR1071 Nt-G3024-TfR1071 Nt-G3018-TfR1071 Nt-G2103-TfR1071 Nt-G3177NTA-TfR1071 Nt-G1018-TfR1071 Ct-G1015-TfR1071 Ct-G1054-TfR1071 Ct-G4068-TfR1071 Ct-G4045-TfR1071 Ct-G1137-TfR1071 Ct-G3008-TfR1071

The results obtained when the antibody concentration was 10 μg/mL are shown in FIGS. 11A to D. FIG. 11A shows the results of evaluating the cell proliferation activity of each antibody on HepG2#P05 cells, and FIGS. 11B to D represent the results of evaluating the cell proliferation activity of each antibody on HepG2#28 cells.

As shown in FIG. 11A , the GPC3-TfR bispecific antibodies all showed more than 20% inhibitory activity. Among them, the inhibitory activity efficiency of 4 antibodies was more than 20% and less than 40%, and the inhibitory activity efficiency of 29 antibodies was more than 40%.

As shown in Figures 11B-D, 20 of the GPC3-TfR bispecific antibodies showed inhibitory activity of more than 20%. Among them, the inhibitory activity efficiency of 10 antibodies was more than 20% and less than 40%, and the inhibitory activity efficiency of 10 antibodies was more than 40%.

To sum up, it is shown that the GPC3-TfR bispecific antibody of the present invention also inhibits cell proliferation for cells whose GPC3 expression is lower than that of HepG2 cells.

[Example 19] Evaluation of cell proliferation inhibitory activity of GPC3-TfR bispecific antibody containing amino acid modified version of anti-TfR antibody TfR1071 By the same method as in Example 5 and Example 6, the following GPC3-TfR bispecific antibody (hereinafter referred to as Nt-G2133- modified TfR1071 bispecific antibody).

Nt-G2133-TfR1071_201 Nt-G2133-TfR1071_202 Nt-G2133-TfR1071_304 Nt-G2133-TfR1071_P98D Nt-G2133-TfR1071_P98E Nt-G2133-TfR1071_P98K

Using the cell viability as an index, the GPC3-TfR bispecific antibodies (six strains) and Nt-G2133-TfR1071 prepared in Example 6 were evaluated against HepG2 cells and HepG2#28 cells by the same method as in Example 9. Proliferation inhibitory activity.

The results obtained when the antibody concentration was 10 μg/mL are shown in FIGS. 12A and B . FIG. 12A shows the results of evaluating the cell proliferation activity of each antibody on HepG2 cells, and FIG. 12B shows the results of evaluating the cell proliferation activity of each antibody on HepG2#28 cells.

As shown in Figure 12A, Nt-G2133-TfR1071_202 among the Nt-G2133-remodeled TfR bispecific antibodies exhibited the same activity as Nt-G2133-TfR1071. As shown in Figure 12B, for cells with low GPC3 expression, Nt-G2133-TfR1071_202 inhibited cell proliferation by more than 50%, showing very high cell proliferation inhibitory activity compared with Nt-G2133-TfR1071.

The above results show that the GPC3-TfR bispecific antibody containing the anti-TfR antibody TfR1071_202 not only has a high GPC3 expression on cells, but also is difficult to exert the cell proliferation inhibitory effect on the GPC3-TfR bispecific antibody containing the anti-TfR antibody TfR1071. The cells with low GPC3 expression have excellent cell proliferation inhibitory activity.

[Example 20] Evaluation of the binding of N-terminal GPC3-TfR1071_202 bispecific antibody to TfR and GPC3 By the same method as Example 5 and Example 6, the following N-terminal GPC3-TfR1071_202 bispecific antibody was produced. Nt-G4025-TfR1071_202 Nt-G2038-TfR1071_202 Nt-G1036-TfR1071_202 Nt-G2002-TfR1071_202 Nt-G1137-TfR1071_202 Nt-G1119-TfR1071_202 Nt-G2133-TfR1071_202 Nt-G2067-TfR1071_202

The binding properties of these N-terminal GPC3-TfR1071_202 bispecific antibodies to human GPC3, monkey GPC3 and human TfR were evaluated by the same method as in Example 7. The results are shown in Figs. 13A to C.

As shown in FIGS. 13A to C, each antibody showed binding to human GPC3, monkey GPC3, and human TfR.

[Example 21] Evaluation of cell proliferation inhibitory activity of N-terminal GPC3-TfR1071_202 bispecific antibody Using the cell survival rate as an index, the GPC3-TfR bispecific antibody (8 strains) prepared in Example 20 was evaluated for its growth inhibitory activity on HepG2 cells and HepG2#28 cells by the same method as in Example 9.

The results obtained when the antibody concentration was 10 μg/mL are shown in FIG. 14 . (A) of FIG. 14 shows the results of the evaluation of the cell proliferation activity of each antibody on HepG2 cells, and (B) shows the results of the evaluation of the cell proliferation activity of each antibody on HepG2#28 cells.

As shown in FIG. 14 , each antibody exhibited GPC3 expression level-dependent cell proliferation inhibitory activity. In addition, it was shown that the degree of inhibition of cell proliferation by each antibody was stronger than that of HN3-GS-TfR1071 on both cells with high GPC3 expression and cells with low GPC3 expression.

[Example 22] Evaluation of the effect of GPC3-TfR bispecific antibody on TfR expression By the same method as in Example 11, the effects of the following N-terminal GPC3-TfR1071 bispecific antibodies (8 types) and GPC3-TfR1071_202 bispecific antibodies (8 types) prepared in Examples 6 and 20 on cell membranes were investigated. The effect of TfR performance above.

Nt-G4025-TfR1071 Nt-G2038-TfR1071 Nt-G1036-TfR1071 Nt-G2002-TfR1071 Nt-G1137-TfR1071 Nt-G1119-TfR1071 Nt-G2133-TfR1071 Nt-G2067-TfR1071 Nt-G4025-TfR1071_202 Nt-G2038-TfR1071_202 Nt-G1036-TfR1071_202 Nt-G2002-TfR1071_202 Nt-G1137-TfR1071_202 Nt-G1119-TfR1071_202 Nt-G2133-TfR1071_202 Nt-G2067-TfR1071_202

By adding 1 μg/mL of each bispecific antibody, each bispecific antibody inhibited the expression of TfR on the cell membrane. It was thus shown that the GPC3-TfR bispecific antibodies of the present invention inhibit cell proliferation by reducing TfR on the cell surface.

[Example 23] Evaluation of the effect of adding ferrous iron on the growth inhibitory activity of GPC3-TfR bispecific antibody It was investigated whether the growth inhibitory activity of the following N-terminal GPC3-TfR1071 bispecific antibodies (8 types) prepared in Example 6 on cancer cells is related to iron.

Nt-G4025-TfR1071 Nt-G2038-TfR1071 Nt-G1036-TfR1071 Nt-G2002-TfR1071 Nt-G1137-TfR1071 Nt-G1119-TfR1071 Nt-G2133-TfR1071 Nt-G2067-TfR1071

Using the cell survival rate as an index, the growth inhibitory activity of the GPC3-TfR bispecific antibody on HepG2 cells and HepG2#28 cells was evaluated by the same method as in Example 9. Further, the conditions also include adding ferric ammonium sulfate (Nacalai, hereinafter referred to as Fe) at a final concentration of 2.5 μM together with the test antibody.

The results obtained when the antibody concentration was 10 μg/mL are shown in FIGS. 15A to D. FIG. Figures 15A and B show the evaluation results of the cell proliferation activity of each antibody on HepG2 cells with and without Fc addition, and Figures 15C and D show the evaluation of the cell proliferation activity of each antibody on HepG2#28 cells with and without Fc addition result.

As shown in Figs. 15A to D, each of the cloned lines lost the cell growth inhibitory activity due to the addition of Fe. By adding Fe, iron can be supplied into cells via a non-TfR-mediated route. Therefore, it is considered that under the condition of adding Fc, the iron-depleted state caused by the inhibition of TfR mediated by the TfR neutralizing antibody TfR435 or the decomposition of TfR mediated by the GPC3-TfR bispecific antibody is released, and the inhibition of proliferation caused by TfR is lost. active. Therefore, it was shown that the GPC3-TfR bispecific antibody of the present invention inhibited cell proliferation by inducing iron depletion.

The present invention has been described in detail with reference to the specific embodiments, and it should be understood by those skilled in the art that various changes and modifications can be added without departing from the spirit and scope of the present invention. This case is based on the Japanese patent application filed on June 26, 2020 (Japanese Patent Application No. 2020-110768), the full text of which is incorporated by reference. [Sequence Listing Non-Key Text]

SEQ ID NO: 1: Base sequence of human TfR extracellular region SEQ ID NO: 2: Amino acid sequence of human TfR extracellular region SEQ ID NO: 3: Base sequence of extracellular region of monkey TfR SEQ ID NO: 4: Amino acid sequence of extracellular region of monkey TfR SEQ ID NO: 5: Base sequence of human TfR SEQ ID NO: 6: Amino acid sequence of human TfR SEQ ID NO: 7: Base sequence of cynomolgus monkey TfR SEQ ID NO: 8: amino acid sequence of cynomolgus monkey TfR SEQ ID NO: 9: Base sequence of human GPC3-mFc SEQ ID NO: 10: Amino acid sequence of human GPC3-mFc SEQ ID NO: 11: Base sequence of mouse GPC3-mFc SEQ ID NO: 12: amino acid sequence of mouse GPC3-mFc SEQ ID NO: 13: Base sequence of human GPC3-rFc SEQ ID NO: 14: Amino acid sequence of human GPC3-rFc SEQ ID NO: 15: Base sequence of mouse GPC3-rFc SEQ ID NO: 16: amino acid sequence of mouse GPC3-rFc SEQ ID NO: 17: Amino acid sequence of soluble human GPC3 SEQ ID NO: 18: Amino acid sequence of human GPC3-GST SEQ ID NO: 19: Base sequence of human GPC3-GST SEQ ID NO: 20: Base sequence of human GPC3-AA-hFc SEQ ID NO: 21: Amino acid sequence of human GPC3-AA-hFc SEQ ID NO: 22: Base sequence of A27 SEQ ID NO: 23: Amino acid sequence of A27 SEQ ID NO: 24: Amino acid sequence of A27 CDR1 SEQ ID NO: 25: Amino acid sequence of A27 CDR2 SEQ ID NO: 26: Amino acid sequence of A27 CDR3 SEQ ID NO: 27: Amino acid sequence of TfR1071 VH SEQ ID NO: 28: Amino acid sequence of TfR1071 HCDR1 SEQ ID NO: 29: Amino acid sequence of TfR1071 HCDR2 SEQ ID NO: 30: Amino acid sequence of TfR1071 HCDR3 SEQ ID NO: 31: Amino acid sequence of TfR1007 VH SEQ ID NO: 32: Amino acid sequence of TfR1007 HCDR1 SEQ ID NO: 33: Amino acid sequence of TfR1007 HCDR2 SEQ ID NO: 34: Amino acid sequence of TfR1007 HCDR3 SEQ ID NO: 35: Amino acid sequence of TfR1071 VH from EVQL SEQ ID NO: 36: Amino acid sequence of TfR1071_201 VH SEQ ID NO: 37: Amino acid sequence of TfR1071_201 HCDR1 SEQ ID NO: 38: Amino acid sequence of TfR1071_201 HCDR2 SEQ ID NO: 39: Amino acid sequence of TfR1071_201 HCDR3 SEQ ID NO: 40: Amino acid sequence of TfR1071_202 VH SEQ ID NO: 41: Amino acid sequence of TfR1071_202 HCDR1 SEQ ID NO: 42: Amino acid sequence of TfR1071_202 HCDR2 SEQ ID NO: 43: Amino acid sequence of TfR1071_202 HCDR3 SEQ ID NO: 44: Amino acid sequence of TfR1071_203 VH SEQ ID NO: 45: Amino acid sequence of TfR1071_203 HCDR1 SEQ ID NO: 46: Amino acid sequence of TfR1071_203 HCDR2 SEQ ID NO: 47: Amino acid sequence of TfR1071_203 HCDR3 SEQ ID NO: 48: Amino acid sequence of TfR1071_204 VH SEQ ID NO: 49: Amino acid sequence of TfR1071_204 HCDR1 SEQ ID NO: 50: Amino acid sequence of TfR1071_204 HCDR2 SEQ ID NO: 51: Amino acid sequence of TfR1071_204 HCDR3 SEQ ID NO: 52: Amino acid sequence of TfR1071_301 VH SEQ ID NO: 53: Amino acid sequence of TfR1071_301 HCDR1 SEQ ID NO: 54: Amino acid sequence of TfR1071_301 HCDR2 SEQ ID NO: 55: Amino acid sequence of TfR1071_301 HCDR3 SEQ ID NO: 56: Amino acid sequence of TfR1071_302 VH SEQ ID NO: 57: Amino acid sequence of TfR1071_302 HCDR1 SEQ ID NO: 58: Amino acid sequence of TfR1071_302 HCDR2 SEQ ID NO: 59: Amino acid sequence of TfR1071_302 HCDR3 SEQ ID NO: 60: Amino acid sequence of TfR1071_303 VH SEQ ID NO: 61: Amino acid sequence of TfR1071_303 HCDR1 SEQ ID NO: 62: Amino acid sequence of TfR1071_303 HCDR2 SEQ ID NO: 63: Amino acid sequence of TfR1071_303 HCDR3 SEQ ID NO: 64: Amino acid sequence of TfR1071_304 VH SEQ ID NO: 65: Amino acid sequence of TfR1071_304 HCDR1 SEQ ID NO: 66: Amino acid sequence of TfR1071_304 HCDR2 SEQ ID NO: 67: Amino acid sequence of TfR1071_304 HCDR3 SEQ ID NO: 68: Amino acid sequence of TfR1071_305 VH SEQ ID NO: 69: Amino acid sequence of TfR1071_305 HCDR1 SEQ ID NO: 70: Amino acid sequence of TfR1071_305 HCDR2 SEQ ID NO: 71: Amino acid sequence of TfR1071_305 HCDR3 SEQ ID NO: 72: Amino acid sequence of TfR1071_306 VH SEQ ID NO: 73: Amino acid sequence of TfR1071_306 HCDR1 SEQ ID NO: 74: Amino acid sequence of TfR1071_306 HCDR2 SEQ ID NO: 75: Amino acid sequence of TfR1071_306 HCDR3 SEQ ID NO: 76: Amino acid sequence of TfR435 VH SEQ ID NO: 77: Amino acid sequence of TfR435 VL SEQ ID NO: 78: Amino acid sequence of TfR1071_P98D VH SEQ ID NO: 79: Amino acid sequence of TfR1071_P98D HCDR1 SEQ ID NO: 80: Amino acid sequence of TfR1071_P98D HCDR2 SEQ ID NO: 81: Amino acid sequence of TfR1071_P98D HCDR3 SEQ ID NO: 82: Amino acid sequence of TfR1071_P98E VH SEQ ID NO: 83: Amino acid sequence of TfR1071_P98E HCDR1 SEQ ID NO: 84: Amino acid sequence of TfR1071_P98E HCDR2 SEQ ID NO: 85: Amino acid sequence of TfR1071_P98E HCDR3 SEQ ID NO: 86: Amino acid sequence of TfR1071_P98K VH SEQ ID NO: 87: Amino acid sequence of TfR1071_P98K HCDR1 SEQ ID NO: 88: Amino acid sequence of TfR1071_P98K HCDR2 SEQ ID NO: 89: Amino acid sequence of TfR1071_P98K HCDR3 SEQ ID NO: 90: Amino acid sequence of G1015 VH SEQ ID NO: 91: Amino acid sequence of G1015 HCDR1 SEQ ID NO: 92: Amino acid sequence of G1015 HCDR2 SEQ ID NO: 93: Amino acid sequence of G1015 HCDR3 SEQ ID NO: 94: Amino acid sequence of G2002 VH SEQ ID NO: 95: Amino acid sequence of G2002 HCDR1 SEQ ID NO: 96: Amino acid sequence of G2002 HCDR2 SEQ ID NO: 97: Amino acid sequence of G2002 HCDR3 SEQ ID NO: 98: Amino acid sequence of G1036 VH SEQ ID NO: 99: Amino acid sequence of G1036 HCDR1 SEQ ID NO: 100: Amino acid sequence of G1036 HCDR2 SEQ ID NO: 101: Amino acid sequence of G1036 HCDR3 SEQ ID NO: 102: Amino acid sequence of G2133 VH SEQ ID NO: 103: Amino acid sequence of G2133 HCDR1 SEQ ID NO: 104: Amino acid sequence of G2133 HCDR2 SEQ ID NO: 105: Amino acid sequence of G2133 HCDR3 SEQ ID NO: 106: Amino acid sequence of G2067 VH SEQ ID NO: 107: Amino acid sequence of G2067 HCDR1 SEQ ID NO: 108: Amino acid sequence of G2067 HCDR2 SEQ ID NO: 109: Amino acid sequence of G2067 HCDR3 SEQ ID NO: 110: Amino acid sequence of G2103 VH SEQ ID NO: 111: Amino acid sequence of G2103 HCDR1 SEQ ID NO: 112: Amino acid sequence of G2103 HCDR2 SEQ ID NO: 113: Amino acid sequence of G2103 HCDR3 SEQ ID NO: 114: Amino acid sequence of G1119 VH SEQ ID NO: 115: Amino acid sequence of G1119 HCDR1 SEQ ID NO: 116: Amino acid sequence of G1119 HCDR2 SEQ ID NO: 117: Amino acid sequence of G1119 HCDR3 SEQ ID NO: 118: Amino acid sequence of G3005 VH SEQ ID NO: 119: Amino acid sequence of G3005 HCDR1 SEQ ID NO: 120: Amino acid sequence of G3005 HCDR2 SEQ ID NO: 121: Amino acid sequence of G3005 HCDR3 SEQ ID NO: 122: Amino acid sequence of G1016 VH SEQ ID NO: 123: Amino acid sequence of G1016 HCDR1 SEQ ID NO: 124: Amino acid sequence of G1016 HCDR2 SEQ ID NO: 125: Amino acid sequence of G1016 HCDR3 SEQ ID NO: 126: Amino acid sequence of G1054 VH SEQ ID NO: 127: Amino acid sequence of G1054 HCDR1 SEQ ID NO: 128: Amino acid sequence of G1054 HCDR2 SEQ ID NO: 129: Amino acid sequence of G1054 HCDR3 SEQ ID NO: 130: Amino acid sequence of G2038 VH SEQ ID NO: 131: Amino acid sequence of G2038 HCDR1 SEQ ID NO: 132: Amino acid sequence of G2038 HCDR2 SEQ ID NO: 133: Amino acid sequence of G2038 HCDR3 SEQ ID NO: 134: Amino acid sequence of G1018 VH SEQ ID NO: 135: Amino acid sequence of G1018 HCDR1 SEQ ID NO: 136: Amino acid sequence of G1018 HCDR2 SEQ ID NO: 137: Amino acid sequence of G1018 HCDR3 SEQ ID NO: 138: Amino acid sequence of G2072 VH SEQ ID NO: 139: Amino acid sequence of G2072 HCDR1 SEQ ID NO: 140: Amino acid sequence of G2072 HCDR2 SEQ ID NO: 141: Amino acid sequence of G2072 HCDR3 SEQ ID NO: 142: Amino acid sequence of G4068 VH SEQ ID NO: 143: Amino acid sequence of G4068 HCDR1 SEQ ID NO: 144: Amino acid sequence of G4068 HCDR2 SEQ ID NO: 145: Amino acid sequence of G4068 HCDR3 SEQ ID NO: 146: Amino acid sequence of G4045 VH SEQ ID NO: 147: Amino acid sequence of G4045 HCDR1 SEQ ID NO: 148: Amino acid sequence of G4045 HCDR2 SEQ ID NO: 149: Amino acid sequence of G4045 HCDR3 SEQ ID NO: 150: Amino acid sequence of G1104 VH SEQ ID NO: 151: Amino acid sequence of G1104 HCDR1 SEQ ID NO: 152: Amino acid sequence of G1104 HCDR2 SEQ ID NO: 153: Amino acid sequence of G1104 HCDR3 SEQ ID NO: 154: Amino acid sequence of G3117 VH SEQ ID NO: 155: Amino acid sequence of G3117 HCDR1 SEQ ID NO: 156: Amino acid sequence of G3117 HCDR2 SEQ ID NO: 157: Amino acid sequence of G3117 HCDR3 SEQ ID NO: 158: Amino acid sequence of G3024 VH SEQ ID NO: 159: Amino acid sequence of G3024 HCDR1 SEQ ID NO: 160: Amino acid sequence of G3024 HCDR2 SEQ ID NO: 161: Amino acid sequence of G3024 HCDR3 SEQ ID NO: 162: Amino acid sequence of G3018 VH SEQ ID NO: 163: Amino acid sequence of G3018 HCDR1 SEQ ID NO: 164: Amino acid sequence of G3018 HCDR2 SEQ ID NO: 165: Amino acid sequence of G3018 HCDR3 SEQ ID NO: 166: Amino acid sequence of G3062 VH SEQ ID NO: 167: Amino acid sequence of G3062 HCDR1 SEQ ID NO: 168: Amino acid sequence of G3062 HCDR2 SEQ ID NO: 169: Amino acid sequence of G3062 HCDR3 SEQ ID NO: 170: Amino acid sequence of G3177 VH SEQ ID NO: 171: Amino acid sequence of G3177 HCDR1 SEQ ID NO: 172: Amino acid sequence of G3177 HCDR2 SEQ ID NO: 173: Amino acid sequence of G3177 HCDR3 SEQ ID NO: 174: Amino acid sequence of G2024 VH SEQ ID NO: 175: Amino acid sequence of G2024 HCDR1 SEQ ID NO: 176: Amino acid sequence of G2024 HCDR2 SEQ ID NO: 177: Amino acid sequence of G2024 HCDR3 SEQ ID NO: 178: Amino acid sequence of G1137 VH SEQ ID NO: 179: Amino acid sequence of G1137 HCDR1 SEQ ID NO: 180: Amino acid sequence of G1137 HCDR2 SEQ ID NO: 181: Amino acid sequence of G1137 HCDR3 SEQ ID NO: 182: Amino acid sequence of G2017 VH SEQ ID NO: 183: Amino acid sequence of G2017 HCDR1 SEQ ID NO: 184: Amino acid sequence of G2017 HCDR2 SEQ ID NO: 185: Amino acid sequence of G2017 HCDR3 SEQ ID NO: 186: Amino acid sequence of G1107 VH SEQ ID NO: 187: Amino acid sequence of G1107 HCDR1 SEQ ID NO: 188: Amino acid sequence of G1107 HCDR2 SEQ ID NO: 189: Amino acid sequence of G1107 HCDR3 SEQ ID NO: 190: Amino acid sequence of G4025 VH SEQ ID NO: 191: Amino acid sequence of G4025 HCDR1 SEQ ID NO: 192: Amino acid sequence of G4025 HCDR2 SEQ ID NO: 193: Amino acid sequence of G4025 HCDR3 SEQ ID NO: 194: Amino acid sequence of G4009 VH SEQ ID NO: 195: Amino acid sequence of G4009 HCDR1 SEQ ID NO: 196: Amino acid sequence of G4009 HCDR2 SEQ ID NO: 197: Amino acid sequence of G4009 HCDR3 SEQ ID NO: 198: Amino acid sequence of G3008 VH SEQ ID NO: 199: Amino acid sequence of G3008 HCDR1 SEQ ID NO: 200: Amino acid sequence of G3008 HCDR2 SEQ ID NO: 201: Amino acid sequence of G3008 HCDR3 SEQ ID NO: 202: Amino acid sequence of G1048 VH SEQ ID NO: 203: Amino acid sequence of G1048 HCDR1 SEQ ID NO: 204: Amino acid sequence of G1048 HCDR2 SEQ ID NO: 205: Amino acid sequence of G1048 HCDR3 SEQ ID NO: 206: Amino acid sequence of G3042 VH SEQ ID NO: 207: Amino acid sequence of G3042 HCDR1 SEQ ID NO: 208: Amino acid sequence of G3042 HCDR2 SEQ ID NO: 209: Amino acid sequence of G3042 HCDR3 SEQ ID NO: 210: Amino acid sequence of G3033 VH SEQ ID NO: 211: Amino acid sequence of G3033 HCDR1 SEQ ID NO: 212: Amino acid sequence of G3033 HCDR2 SEQ ID NO: 213: Amino acid sequence of G3033 HCDR3 SEQ ID NO: 214: Amino acid sequence of G4037 VH SEQ ID NO: 215: Amino acid sequence of G4037 HCDR1 SEQ ID NO: 216: Amino acid sequence of G4037 HCDR2 SEQ ID NO: 217: Amino acid sequence of G4037 HCDR3 SEQ ID NO: 218: Amino acid sequence of G1122 VH SEQ ID NO: 219: Amino acid sequence of G1122 HCDR1 SEQ ID NO: 220: Amino acid sequence of G1122 HCDR2 SEQ ID NO: 221: Amino acid sequence of G1122 HCDR3 SEQ ID NO: 222: Amino acid sequence of G2011 VH SEQ ID NO: 223: Amino acid sequence of G2011 HCDR1 SEQ ID NO: 224: Amino acid sequence of G2011 HCDR2 SEQ ID NO: 225: Amino acid sequence of G2011 HCDR3 SEQ ID NO: 226: Amino acid sequence of G2025 VH SEQ ID NO: 227: Amino acid sequence of G2025 HCDR1 SEQ ID NO: 228: Amino acid sequence of G2025 HCDR2 SEQ ID NO: 229: Amino acid sequence of G2025 HCDR3 SEQ ID NO: 230: Amino acid sequence of G4033 VH SEQ ID NO: 231: Amino acid sequence of G4033 HCDR1 SEQ ID NO: 232: Amino acid sequence of G4033 HCDR2 SEQ ID NO: 233: Amino acid sequence of G4033 HCDR3 SEQ ID NO: 234: Amino acid sequence of G3062_NYA VH SEQ ID NO: 235: Amino acid sequence of G3062_NYA HCDR1 SEQ ID NO: 236: Amino acid sequence of G3062_NYA HCDR2 SEQ ID NO: 237: Amino acid sequence of G3062_NYA HCDR3 SEQ ID NO: 238: Amino acid sequence of G3177_NTA VH SEQ ID NO: 239: Amino acid sequence of G3177_NTA HCDR1 SEQ ID NO: 240: Amino acid sequence of G3177_NTA HCDR2 SEQ ID NO: 241: Amino acid sequence of G3177_NTA HCDR3 SEQ ID NO: 242: Amino acid sequence of G2024_NNA VH SEQ ID NO: 243: Amino acid sequence of G2024_NNA HCDR1 SEQ ID NO: 244: Amino acid sequence of G2024_NNA HCDR2 SEQ ID NO: 245: Amino acid sequence of G2024_NNA HCDR3 SEQ ID NO: 246: Amino acid sequence of G2017_NNA VH SEQ ID NO: 247: Amino acid sequence of G2017_NNA HCDR1 SEQ ID NO: 248: Amino acid sequence of G2017_NNA HCDR2 SEQ ID NO: 249: Amino acid sequence of G2017_NNA HCDR3 SEQ ID NO: 250: Amino acid sequence of HN3 VH SEQ ID NO: 251: Amino acid sequence of Fc of IgG4PE R409K SEQ ID NO: 252: Base sequence of IgG4 CH1 SEQ ID NO: 253: Amino acid sequence of synthetic structure SEQ ID NO: 254: Base sequence of IgG4PE R409K CH SEQ ID NO: 255: Amino acid sequence of synthetic structure SEQ ID NO: 256: Amino acid sequence of the linker SEQ ID NO: 257: Base sequence of TfR1071 VH from EVQL SEQ ID NO: 258: Base sequence of IgG4 CH1 including stop codon SEQ ID NO: 259: Amino acid sequence of mouse TfR SEQ ID NO: 260: Amino acid sequence of apical domain mouse chimera of human TfR extracellular domain SEQ ID NO: 261: Amino acid sequence of the protease-like domain mouse chimera of the extracellular region of human TfR SEQ ID NO: 262: Amino acid sequence of A02 SEQ ID NO: 263: Amino acid sequence of A03 SEQ ID NO: 264: Amino acid sequence of A04 SEQ ID NO: 265: Amino acid sequence of A05 SEQ ID NO: 266: Amino acid sequence of A07 SEQ ID NO: 267: Amino acid sequence of A14 SEQ ID NO: 268: Amino acid sequence of TfR1071_307 VH SEQ ID NO: 269: Amino acid sequence of TfR1071_307 HCDR1 SEQ ID NO: 270: Amino acid sequence of TfR1071_307 HCDR2 SEQ ID NO: 271: Amino acid sequence of TfR1071_307 HCDR3 SEQ ID NO: 272: Amino acid sequence of CL

Figure 1 shows the structure of the bispecific antibody of the present invention. (A) shows the structure of an N-terminal GPC3-TfR bispecific antibody or an N-terminal TfR-GPC3 bispecific antibody. (B) shows the structure of a C-terminal GPC3-TfR bispecific antibody or a C-terminal TfR-GPC3 bispecific antibody. (C) shows the structure of GPC3-GS-TfR bispecific antibody or TfR-GS-GPC3 bispecific antibody. Figure 2A shows the results of evaluating the binding of each GPC3-TfR bispecific antibody to His-human TfR. The vertical axis represents absorbance, and the horizontal axis represents the name of the antibody used. Anti-DNP antibody was used as a negative control. Figure 2B shows the results of evaluating the binding of each GPC3-TfR bispecific antibody to His-human TfR. The vertical axis represents absorbance, and the horizontal axis represents the name of the antibody used. Anti-DNP antibody was used as a negative control. Figure 2C shows the results of evaluating the binding of each GPC3-TfR bispecific antibody to human GPC3-His. The vertical axis represents absorbance, and the horizontal axis represents the name of the antibody used. Anti-DNP antibody was used as a negative control. Figure 2D shows the results of evaluating the binding of each GPC3-TfR bispecific antibody to human GPC3-His. The vertical axis represents absorbance, and the horizontal axis represents the name of the antibody used. Anti-DNP antibody was used as a negative control. Figure 2E shows the results of evaluating the binding of each GPC3-TfR bispecific antibody to monkey GPC3-His. The vertical axis represents absorbance, and the horizontal axis represents the name of the antibody used. Anti-DNP antibody was used as a negative control. Figure 2F shows the results of evaluating the binding of each GPC3-TfR bispecific antibody to monkey GPC3-His. The vertical axis represents absorbance, and the horizontal axis represents the name of the antibody used. Anti-DNP antibody was used as a negative control. FIG. 3 shows the results of evaluating the amount of antigen expression in liver cancer cells by flow cytometry. (A) shows the result of evaluating the expression level of TfR in HepG2 cells by flow cytometry, and (B) shows the result of evaluating the expression level of TfR in HLE cells by flow cytometry. (C) shows the result of evaluating the expression level of GPC3 in HepG2 cells by flow cytometry, and (D) shows the result of evaluating the expression level of GPC3 in HLE cells by flow cytometry. The vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity. The white histogram represents the binding of anti-TfR antibody or anti-GPC3 antibody, and the gray histogram represents the binding of the negative control anti-DNP antibody. FIG. 4 shows the results of evaluating the growth inhibitory activity of each anti-TfR antibody on hepatoma cells. (A) shows the results of evaluating the growth inhibitory activity of anti-TfR antibodies on HLE cells, and (B) shows the results of evaluating the growth inhibitory activity of anti-TfR antibodies on HepG2 cells. The vertical axis represents the cell viability relative to the control group. FIG. 5A shows the results of evaluating the growth inhibitory activity of GPC3-TfR bispecific antibody on hepatoma cells (HLE cells). The vertical axis represents the cell viability relative to the control group. Figure 5B shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibody on hepatoma cells (HLE cells). The vertical axis represents the cell viability relative to the control group. Figure 5C shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibody on hepatoma cells (HLE cells). The vertical axis represents the cell viability relative to the control group. Figure 5D shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibody on hepatoma cells (HepG2 cells). The vertical axis represents the cell viability relative to the control group. Figure 5E shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibody on hepatoma cells (HepG2 cells). The vertical axis represents the cell viability relative to the control group. Figure 5F shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibody on hepatoma cells (HepG2 cells). The vertical axis represents the cell viability relative to the control group. Fig. 6 shows the results of quantification of the number of expression of TfR on the cell surface when HN3-GS-TfR1071 was added to HepG2 cells by flow cytometry. The vertical axis represents the average number of TfR molecules per cell on the cell surface. Figure 7 shows the results of evaluating the competition between each anti-GPC3 antibody and a known anti-GPC3 antibody for binding to human GPC3. (A) shows the results of evaluating the binding of anti-GPC3 antibody (test antibody) to GPC3-AA-Fc in the presence or absence of the competing antibody GC33 by ELISA. The vertical axis represents the absorbance, the white bar represents the absorbance of each tested antibody in the absence of GC33, and the black bar represents the absorbance of each tested antibody in the presence of GC33. The vertical axis of (B) represents the relative value (%) of the absorbance of each test antibody in the presence of GC33 relative to the absorbance of each test antibody in the absence of GC33. NA means unable to evaluate. FIG. 8 shows a list of C-terminal GPC3-TfR bispecific antibodies prepared in Example 14. FIG. 9A shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14. FIG. 9B shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14. FIG. FIG. 9C shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14. FIG. FIG. 9D shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14. FIG. FIG. 9E shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14. FIG. FIG. 9F shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14. FIG. FIG. 9G shows a list of N-terminal GPC3-TfR bispecific antibodies prepared in Example 14. FIG. Figure 10 shows the results of evaluating the expression levels of GPC3 and TfR in GPC3 knockdown HepG2 cells. Figure 11A shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibodies on GPC3 knockdown HepG2 cells (HepG2#P05 cells, GPC3++). Figure 11B shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibodies on GPC3 knockdown HepG2 cells (HepG2#28 cells, GPC3+). Figure 11C shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibodies on GPC3 knockdown HepG2 cells (HepG2#28 cells, GPC3+). Figure 11D shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibodies on GPC3 knockdown HepG2 cells (HepG2#28 cells, GPC3+). Figure 12A shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibody on HepG2 cells (GPC3+++). Figure 12B shows the results of evaluating the proliferation inhibitory activity of GPC3-TfR bispecific antibody on HepG2#28 cells (GPC3+). Figure 13A shows the results of evaluating the binding of GPC3-TfR bispecific antibodies to human GPC3. Figure 13B shows the results of evaluating the binding of GPC3-TfR bispecific antibodies to monkey GPC3. Figure 13C shows the results of evaluating the binding of GPC3-TfR bispecific antibodies to human TfR. Figure 14 shows the results of evaluating the cell proliferation inhibitory activity of the GPC3-TfR bispecific antibody. (A) shows the growth inhibitory activity of GPC3-TfR bispecific antibody on HepG2 cells (GPC3+++). (B) shows the growth inhibitory activity of GPC3-TfR bispecific antibody on HepG2#28 cells. Figure 15A shows the results of evaluating the effect of adding ferrous iron on the proliferation inhibitory activity of the GPC3-TfR bispecific antibody on HepG2 cells. Figure 15B shows the results of evaluating the effect of adding ferrous iron on the proliferation inhibitory activity of the GPC3-TfR bispecific antibody on HepG2 cells. Figure 15C shows the results of evaluating the effect of the addition of ferrous iron on the proliferation inhibitory activity of the GPC3-TfR bispecific antibody on HepG2#28 cells. Figure 15D shows the results of evaluating the effect of adding ferrous iron on the proliferation inhibitory activity of the GPC3-TfR bispecific antibody on HepG2#28 cells.

         
           <![CDATA[ <110> Kyowa Kirin Co., Ltd.]]>
           <![CDATA[ <120> Bispecific antibody binding to GPC3 and TfR]]>
           <![CDATA[ <130> W531973]]>
           <![CDATA[ <150> JP2020-110768]]>
           <![CDATA[ <151> 2020-06-26]]>
           <![CDATA[ <160> 272 ]]>
           <![CDATA[ <170> PatentIn version 3.5]]>
           <![CDATA[ <210> 1]]>
           <![CDATA[ <211> 2016]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 1]]>
          tgtaaagggg tagaaccaaa aactgagtgt gagagactgg caggaaccga gtctccagtg 60
          agggaggagc caggagagga cttccctgca gcacgtcgct tatattggga tgacctgaag 120
          agaaagttgt cggagaaact ggacagcaca gacttcaccg gcaccatcaa gctgctgaat 180
          gaaaattcat atgtccctcg tgaggctgga tctcaaaaag atgaaaatct tgcgttgtat 240
          gttgaaaatc aatttcgtga atttaaactc agcaaagtct ggcgtgatca acattttgtt 300
          aagattcagg tcaaagacag cgctcaaaac tcggtgatca tagttgataa gaacggtaga 360
          cttgtttacc tggtggagaa tcctgggggt tatgtggcgt atagtaaggc tgcaacagtt 420
          actggtaaac tggtccatgc taattttggt actaaaaaag attttgagga tttatacact 480
          cctgtgaatg gatctatagt gattgtcaga gcagggaaaa tcacctttgc agaaaaggtt 540
          gcaaatgctg aaagcttaaa tgcaattggt gtgttgatat acatggacca gactaaattt 600
          cccattgtta acgcagaact ttcattcttt ggacatgctc atctggggac aggtgaccct 660
          tacacacctg gattcccttc cttcaatcac actcagtttc caccatctcg gtcatcagga 720
          ttgcctaata tacctgtcca gacaatctcc agagctgctg cagaaaagct gtttgggaat 780
          atggaaggag actgtccctc tgactggaaa acagactcta catgtaggat ggtaacctca 840
          gaaagcaaga atgtgaagct cactgtgagc aatgtgctga aagagataaa aattcttaac 900
          atctttggag ttattaaagg ctttgtagaa ccagatcact atgttgtagt tggggcccag 960
          agagatgcat ggggccctgg agctgcaaaa tccggtgtag gcacagctct cctattgaaa 1020
          cttgcccaga tgttctcaga tatggtctta aaagatgggt ttcagcccag cagaagcatt 1080
          atctttgcca gttggagtgc tggagacttt ggatcggttg gtgccactga atggctagg 1140
          ggataccttt cgtccctgca tttaaaggct ttcacttata ttaatctgga taaagcggtt 1200
          cttggaacca gcaacttcaa ggtttctgcc agcccactgt tgtatacgct tattgagaaa 1260
          acaatgcaaa atgtgaagca tccggttact gggcaatttc tatatcagga cagcaactgg 1320
          gccagcaaag ttgagaaact cactttagac aatgctgctt tccctttcct tgcatattct 1380
          ggaatcccag cagtttcttt ctgtttttgc gaggacacag attatcctta tttgggaacc 1440
          accatggaca cctataagga actgattgag aggattcctg agttgaacaa agtggcacga 1500
          gcagctgcag aggtcgctgg tcagttcgtg attaaactaa cccatgatgt tgaattgaac 1560
          ctggactatg agaggtacaa cagccaactg ctttcatttg tgagggatct gaaccaatac 1620
          agagcagaca taaaggaaat gggcctgagt ttacagtggc tgtattctgc tcgtggagac 1680
          ttcttccgtg ctacttccag actaacaaca gatttcggga atgctgagaa aacagacaga 1740
          tttgtcatga agaaactcaa tgatcgtgtc atgagagtgg agtatcactt cctctctccc 1800
          tacgtatctc caaaagagtc tcctttccga catgtcttct ggggctccgg ctctcacacg 1860
          ctgccagctt tactggagaa cttgaaactg cgtaaacaaa ataacggtgc ttttaatgaa 1920
          acgctgttca gaaaccagtt ggctctagct acttggacta ttcagggagc tgcaaatgcc 1980
          ctctctggtg acgtttggga cattgacaat gagttt 2016
           <![CDATA[ <210> 2]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 2]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
                      100 105 110
          Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro
                  115 120 125
          Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr
          145 150 155 160
          Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser
                  195 200 205
          Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp
                      260 265 270
          Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr
                  275 280 285
          Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
                      420 425 430
          Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
                          565 570 575
          Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
              610 615 620
          Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 3]]>
           <![CDATA[ <211> 2016]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Monkey]]>
           <![CDATA[ <400> 3]]>
          tgtaaagggg tagaaccaaa aactgagtgt gagagactgg caggcaccga gtctccagcg 60
          agggaggagc cagaagagga cttccctgcg gcaccgcgct tatactggga cgacctgaag 120
          agaaagttgt cagagaaact ggacaccaca gacttcacca gcaccatcaa gctgctgaat 180
          gaaaatttat atgtccctcg tgaggctgga tctcaaaaag atgaaaatct tgcattgtat 240
          attgaaaatc aatttcgtga atttaaacta agcaaagtct ggcgtgatca acattttgtt 300
          aagattcagg tcaaggacag tgctcaaaac tcggtgatca tagttgataa gaatggtgga 360
          cttgtttacc tggtggagaa tcctgggggt tatgtggcat atagtaaggc tgcaacagtt 420
          actggtaaac tggtccatgc taattttggt actaaaaaag actttgagga tttagactct 480
          cctgtgaatg gatctatagt gattgtcaga gcaggaaaaa tcacctttgc agaaaaggtt 540
          gcaaatgctg aaagcttaaa tgcaattggt gtcttgatat atatggacca gactaaattt 600
          cctattgtta aggcagacct ttcattcttt ggacatgctc atctgggaac aggtgaccct 660
          tacacacctg gattcccttc cttcaatcac actcagtttc caccatctca gtcgtcagga 720
          ttgcctaata tacctgtcca aacgatctcc agagctgctg cagaaaagct gtttggaaat 780
          atggagggag actgtccctc tgactggaaa acagactcta cgtgtaagat ggtaacctcg 840
          gaaaacaaga gtgtgaagct cacggtgagc aatgtgctga aagagacaaa aattcttaac 900
          atctttggag ttattaaagg cttcgtagaa ccagatcact atgttgtggt tggggcccag 960
          agagatgcgt ggggccccgg agctgcaaaa tccagtgtgg ggacagctct cctgttgaaa 1020
          cttgcccaga tgttctcaga tatggtctta aaagatgggt ttcagcccag cagaagcatt 1080
          atctttgcca gttggagtgc tggagacttt ggatcggttg gtgccactga atggctagg 1140
          ggataccttt catccttgca tttaaaggct ttcacttaca ttaatctgga taaagcggtg 1200
          cttggaacca gcaacttcaa ggtttctgcc agcccgttgt tgtatacgct gattgagaaa 1260
          acaatgcaag atgtgaaaca tccggttact gggcgatctc tatatcagga cagcaactgg 1320
          gccagcaaag ttgagaaact cactttagac aatgctgctt tccctttcct tgcgtattct 1380
          ggaatcccag cagtttcttt ctgtttttgt gaggacacag attatcctta cttgggcacc 1440
          accatggaca cctataagga actggtggag aggattcctg agctgaacaa agtggcacga 1500
          gcagcggcag aagtagctgg tcagttcgtg attaaactga cccatgatac tgaattgaac 1560
          ctggactatg agaggtacaa cagccagctg cttttgtttt tgagggatct gaaccagtac 1620
          agagcagatg taaaggaaat gggcctgagc ttgcagtggc tgtattctgc tcgtggagac 1680
          tttttccgtg ctacttccag gctaacaaca gatttcagga atgctgagaa aagggacaag 1740
          tttgtcatga agaagctcaa tgatcgtgtc atgagagtcg agtattactt cctctcaccc 1800
          tatgtgtctc caaaagagtc tcctttccgg cacgtcttct ggggctcggg ctcccacacg 1860
          ctgtccgctt tactggagag cttgaaactg cgtagacaga ataacagtgc ttttaatgaa 1920
          acgctgttca gaaaccagct ggctctcgcg acttggacta ttcagggagc tgcaaatgcc 1980
          ctttctggtg acgtttggga cattgacaat gagttt 2016
           <![CDATA[ <210> 4]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Monkey]]>
           <![CDATA[ <400> 4]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Ala Arg Glu Glu Pro Glu Glu Asp Phe Pro Ala Ala Pro
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Thr Thr Asp Phe Thr Ser Thr Ile Lys Leu Leu Asn Glu Asn Leu Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Ile Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
                      100 105 110
          Ile Ile Val Asp Lys Asn Gly Gly Leu Val Tyr Leu Val Glu Asn Pro
                  115 120 125
          Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Asp Ser
          145 150 155 160
          Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Lys Ala Asp Leu Ser
                  195 200 205
          Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Gln Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp
                      260 265 270
          Ser Thr Cys Lys Met Val Thr Ser Glu Asn Lys Ser Val Lys Leu Thr
                  275 280 285
          Val Ser Asn Val Leu Lys Glu Thr Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Ser Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asp Val Lys His Pro Val Thr Gly Arg
                      420 425 430
          Ser Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Val Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Thr Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Leu Phe Leu Arg Asp Leu Asn Gln Tyr Arg Ala Asp Val
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Arg Asn Ala Glu
                          565 570 575
          Lys Arg Asp Lys Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr Tyr Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Ser Ala Leu
              610 615 620
          Leu Glu Ser Leu Lys Leu Arg Arg Gln Asn Asn Ser Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 5]]>
           <![CDATA[ <211> 2280]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 5]]>
          atgatggatc aagctagatc agcattctct aacttgtttg gtggagaacc attgtcatat 60
          acccggttca gcctggctcg gcaagtagat ggcgataaca gtcatgtgga gatgaaactt 120
          gctgtagatg aagaagaaaa tgctgacaat aacacaaagg ccaatgtcac aaaaccaaaa 180
          aggtgtagtg gaagtatctg ctatgggact attgctgtga tcgtcttttt cttgattgga 240
          tttatgattg gctacttggg ctattgtaaa ggggtagaac caaaaactga gtgtgagaga 300
          ctggcaggaa ccgagtctcc agtgagggag gagccaggag aggacttccc tgcagcacgt 360
          cgcttatatt gggatgacct gaagagaaag ttgtcggaga aactggacag cacagacttc 420
          accggcacca tcaagctgct gaatgaaaat tcatatgtcc ctcgtgaggc tggatctcaa 480
          aaagatgaaa atcttgcgtt gtatgttgaa aatcaatttc gtgaatttaa actcagcaaa 540
          gtctggcgtg atcaacattt tgttaagatt caggtcaaag acagcgctca aaactcggtg 600
          atcatagttg ataagaacgg tagacttgtt tacctggtgg agaatcctgg gggttatgtg 660
          gcgtatagta aggctgcaac agttactggt aaactggtcc atgctaattt tggtactaaa 720
          aaagattttg aggatttata cactcctgtg aatggatcta tagtgattgt cagagcaggg 780
          aaaatcacct ttgcagaaaa ggttgcaaat gctgaaagct taaatgcaat tggtgtgttg 840
          atatacatgg accagactaa atttcccatt gttaacgcag aactttcatt ctttggacat 900
          gctcatctgg ggacaggtga cccttacaca cctggattcc cttccttcaa tcacactcag 960
          tttccaccat ctcggtcatc aggattgcct aatatacctg tccagacaat ctccagagct 1020
          gctgcagaaa agctgtttgg gaatatggaa ggagactgtc cctctgactg gaaaacagac 1080
          tctacatgta ggatggtaac ctcagaaagc aagaatgtga agctcactgt gagcaatgtg 1140
          ctgaaagaga taaaaattct taacatcttt ggagttatta aaggctttgt agaaccagat 1200
          cactatgttg tagttggggc ccagagagat gcatggggcc ctggagctgc aaaatccggt 1260
          gtaggcacag ctctcctatt gaaacttgcc cagatgttct cagatatggt cttaaaagat 1320
          gggtttcagc ccagcagaag cattatcttt gccagttgga gtgctggaga ctttggatcg 1380
          gttggtgcca ctgaatggct agagggatac ctttcgtccc tgcatttaaa ggctttcact 1440
          tatattaatc tggataaagc ggttcttggt accagcaact tcaaggtttc tgccagccca 1500
          ctgttgtata cgcttattga gaaaacaatg caaaatgtga agcatccggt tactgggcaa 1560
          tttctatatc aggacagcaa ctgggccagc aaagttgaga aactcacttt agacaatgct 1620
          gctttccctt tccttgcata ttctggaatc ccagcagttt ctttctgttt ttgcgaggac 1680
          acagattatc cttatttggg taccaccatg gacacctata aggaactgat tgagaggatt 1740
          cctgagttga acaaagtggc acgagcagct gcagaggtcg ctggtcagtt cgtgattaaa 1800
          ctaacccatg atgttgaatt gaacctggac tatgagaggt acaacagcca actgctttca 1860
          tttgtgaggg atctgaacca atacagagca gacataaagg aaatgggcct gagtttacag 1920
          tggctgtatt ctgctcgtgg agacttcttc cgtgctactt ccagactaac aacagatttc 1980
          gggaatgctg agaaaacaga cagatttgtc atgaagaaac tcaatgatcg tgtcatgaga 2040
          gtggagtatc acttcctctc tccctacgta tctccaaaag agtctccttt ccgacatgtc 2100
          ttctggggct ccggctctca cacgctgcca gctttactgg agaacttgaa actgcgtaaa 2160
          caaaataacg gtgcttttaa tgaaacgctg ttcagaaacc agttggctct agctacttgg 2220
          actattcagg gagctgcaaa tgccctctct ggtgacgttt gggacattga caatgagttt 2280
           <![CDATA[ <210> 6]]>
           <![CDATA[ <211> 760]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 6]]>
          Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
          1 5 10 15
          Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
                      20 25 30
          Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala
                  35 40 45
          Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly
              50 55 60
          Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly
          65 70 75 80
          Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
                          85 90 95
          Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro
                      100 105 110
          Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys
                  115 120 125
          Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile
              130 135 140
          Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln
          145 150 155 160
          Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe
                          165 170 175
          Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
                      180 185 190
          Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg
                  195 200 205
          Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
              210 215 220
          Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
          225 230 235 240
          Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile
                          245 250 255
          Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
                      260 265 270
          Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
                  275 280 285
          Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly
              290 295 300
          Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
          305 310 315 320
          Phe Pro Pro Ser Arg Ser Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
                          325 330 335
          Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
                      340 345 350
          Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser
                  355 360 365
          Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile
              370 375 380
          Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
          385 390 395 400
          His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
                          405 410 415
          Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
                      420 425 430
          Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
                  435 440 445
          Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
              450 455 460
          Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
          465 470 475 480
          Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
                          485 490 495
          Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn
                      500 505 510
          Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp
                  515 520 525
          Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
              530 535 540
          Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
          545 550 555 560
          Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
                          565 570 575
          Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
                      580 585 590
          Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn
                  595 600 605
          Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp
              610 615 620
          Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln
          625 630 635 640
          Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
                          645 650 655
          Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys
                      660 665 670
          Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro
                  675 680 685
          Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
              690 695 700
          Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys
          705 710 715 720
          Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
                          725 730 735
          Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
                      740 745 750
          Val Trp Asp Ile Asp Asn Glu Phe
                  755 760
           <![CDATA[ <210> 7]]>
           <![CDATA[ <211> 2280]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Crab-eating Macaque]]>
           <![CDATA[ <400> 7]]>
          atgatggatc aagctagatc agcattctct aacttgtttg gtggagaacc attgtcatat 60
          acccggttca gcctggctcg gcaagtagac ggcgataaca gtcatgtgga gatgaaactt 120
          gctgtagatg atgaagaaaa tgctgacaat aacacaaagg ccaatggcac aaaaccaaaa 180
          aggtgtggtg gaaatatctg ctatgggact attgccgtga tcatcttttt cttgattggg 240
          tttatgattg gctacttggg ctattgtaaa ggggtagaac caaaaactga gtgtgagaga 300
          ctggcaggca ccgagtctcc agcgagggag gagccagaag aggacttccc tgcggcaccg 360
          cgcttatact gggacgacct gaagagaaag ttgtcagaga aactggacac cacagacttc 420
          accagcacca tcaagctgct gaatgaaaat ttatatgtcc ctcgtgaggc tggatctcaa 480
          aaagatgaaa atcttgcatt gtatattgaa aatcaatttc gtgaatttaa actaagcaaa 540
          gtctggcgtg atcaacattt tgttaagatt caggtcaagg acagtgctca aaactcggtg 600
          atcatagttg ataagaatgg tggacttgtt tacctggtgg agaatcctgg gggttatgtg 660
          gcatatagta aggctgcaac agttactggt aaactggtcc atgctaattt tggtactaaa 720
          aaagactttg aggatttaga ctctcctgtg aatggatcta tagtgattgt cagagcagga 780
          aaaatcacct ttgcagaaaa ggttgcaaat gctgaaagct taaatgcaat tggtgtcttg 840
          atatatatgg accagactaa atttcctatt gttaaggcag acctttcatt ctttggacat 900
          gctcatctgg gaacaggtga cccttacaca cctggattcc cttccttcaa tcacactcag 960
          tttccaccat ctcagtcgtc aggattgcct aatatacctg tccaaacgat ctccagagct 1020
          gctgcagaaa agctgtttgg aaatatggag ggagactgtc cctctgactg gaaaacagac 1080
          tctacgtgta agatggtaac ctcggaaaac aagagtgtga agctcacggt gagcaatgtg 1140
          ctgaaagaga caaaaattct taacatcttt ggagttatta aaggcttcgt agaaccagat 1200
          cactatgttg tggttggggc ccagagagat gcgtggggcc ccggagctgc aaaatccagt 1260
          gtggggacag ctctcctgtt gaaacttgcc cagatgttct cagatatggt cttaaaagat 1320
          gggtttcagc ccagcagaag cattatcttt gccagttgga gtgctggaga ctttggatcg 1380
          gttggtgcca ctgaatggct agagggatac ctttcatcct tgcatttaaa ggctttcact 1440
          tacattaatc tggataaagc ggtgcttgga accagcaact tcaaggtttc tgccagcccg 1500
          ttgttgtata cgctgattga gaaaacaatg caagatgtga aacatccggt tactgggcga 1560
          tctctatatc aggacagcaa ctgggccagc aaagttgaga aactcacttt agacaatgct 1620
          gctttccctt tccttgcgta ttctggaatc ccagcagttt ctttctgttt ttgtgaggac 1680
          acagattatc cttacttggg caccaccatg gacacctata aggaactggt ggagaggatt 1740
          cctgagctga acaaagtggc acgagcagcg gcagaagtag ctggtcagtt cgtgattaaa 1800
          ctgacccatg atactgaatt gaacctggac tatgagaggt acaacagcca gctgcttttg 1860
          ttttttgaggg atctgaacca gtacagagca gatgtaaagg aaatgggcct gagcttgcag 1920
          tggctgtatt ctgctcgtgg agactttttc cgtgctactt ccaggctaac aacagatttc 1980
          aggaatgctg agaaaaggga caagtttgtc atgaagaagc tcaatgatcg tgtcatgaga 2040
          gtcgagtatt acttcctctc accctatgtg tctccaaaag agtctccttt ccggcacgtc 2100
          ttctggggct cgggctccca cacgctgtcc gctttactgg agagcttgaa actgcgtaga 2160
          cagaataaca gtgcttttaa tgaaacgctg ttcagaaacc agctggctct cgcgacttgg 2220
          actattcagg gagctgcaaa tgccctttct ggtgacgttt gggacattga caatgagttt 2280
           <![CDATA[ <210> 8]]>
           <![CDATA[ <211> 760]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Crab-eating Macaque]]>
           <![CDATA[ <400> 8]]>
          Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
          1 5 10 15
          Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
                      20 25 30
          Asn Ser His Val Glu Met Lys Leu Ala Val Asp Asp Glu Glu Asn Ala
                  35 40 45
          Asp Asn Asn Thr Lys Ala Asn Gly Thr Lys Pro Lys Arg Cys Gly Gly
              50 55 60
          Asn Ile Cys Tyr Gly Thr Ile Ala Val Ile Ile Phe Phe Leu Ile Gly
          65 70 75 80
          Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
                          85 90 95
          Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu Pro
                      100 105 110
          Glu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu Lys
                  115 120 125
          Arg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr Ile
              130 135 140
          Lys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly Ser Gln
          145 150 155 160
          Lys Asp Glu Asn Leu Ala Leu Tyr Ile Glu Asn Gln Phe Arg Glu Phe
                          165 170 175
          Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
                      180 185 190
          Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly
                  195 200 205
          Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
              210 215 220
          Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
          225 230 235 240
          Lys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile
                          245 250 255
          Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
                      260 265 270
          Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
                  275 280 285
          Pro Ile Val Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu Gly
              290 295 300
          Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
          305 310 315 320
          Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
                          325 330 335
          Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
                      340 345 350
          Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser
                  355 360 365
          Glu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Thr
              370 375 380
          Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
          385 390 395 400
          His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
                          405 410 415
          Ala Lys Ser Ser Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
                      420 425 430
          Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
                  435 440 445
          Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
              450 455 460
          Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
          465 470 475 480
          Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
                          485 490 495
          Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asp
                      500 505 510
          Val Lys His Pro Val Thr Gly Arg Ser Leu Tyr Gln Asp Ser Asn Trp
                  515 520 525
          Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
              530 535 540
          Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
          545 550 555 560
          Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
                          565 570 575
          Val Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
                      580 585 590
          Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu Asn
                  595 600 605
          Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg Asp
              610 615 620
          Leu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly Leu Ser Leu Gln
          625 630 635 640
          Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
                          645 650 655
          Thr Thr Asp Phe Arg Asn Ala Glu Lys Arg Asp Lys Phe Val Met Lys
                      660 665 670
          Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr Tyr Phe Leu Ser Pro
                  675 680 685
          Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
              690 695 700
          Gly Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg Arg
          705 710 715 720
          Gln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
                          725 730 735
          Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
                      740 745 750
          Val Trp Asp Ile Asp Asn Glu Phe
                  755 760
           <![CDATA[ <210> 9]]>
           <![CDATA[ <211> 2376]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 9]]>
          atggccggaa ctgtacggac agcatgtctc gtggtggcca tgctcttgag tctggacttt 60
          cccggccaag cccaacctcc ccctcctccc cccgatgcca cctgccatca ggttaggtca 120
          tttttccaaa ggcttcagcc cgggctgaag tgggttcctg aaacccctgt ccctgggtcc 180
          gatcttcagg tctgcctccc caaagggcct acctgctgtt cccgtaaaat ggaggagaag 240
          taccagttga ccgctagact caacatggag cagctgctcc agtcagcatc tatggagttg 300
          aagttcctga ttatacaaaa cgccgcagtt ttccaggagg catttgaaat tgtagtacgt 360
          cacgcaaaaa actacaccaa tgcaatgttc aagaataact atccctctct gacccctcaa 420
          gcctttgagt tcgtaggcga gttcttcacc gatgtgagtc tctatatcct cgggtcagat 480
          atcaacgtag atgacatggt aaatgaactt tttgacagcc tgtttcccgt tatttatacc 540
          cagctgatga accctggcct tccagactca gcactcgaca taaatgaatg cctgcggggg 600
          gccagacgcg acttgaaggt ttttgggaac tttccaaagc ttatcatgac acaagtttca 660
          aagtctttgc aagtgacccg tattttcctt caagctctca accttggcat tgaggtcata 720
          aataccaccg atcaccttaa gttcagcaaa gactgcggga gaatgctgac tcgcatgtgg 780
          tactgcagct attgccaggg gctgatgatg gtgaagcctt gtggaggcta ttgtaatgtg 840
          gtgatgcagg gatgtatggc aggcgtagta gagatcgaca aatattggcg tgagtacatc 900
          ctctctttgg aggaacttgt aaacggtatg taccgaatat atgacatgga aaacgtgctt 960
          ctgggactct tctcaaccat ccacgactca atccaatatg ttcaaaagaa cgcaggaaaa 1020
          cttactacaa ctattggaaa gctgtgtgca cattcacagc agcgtcagta taggagcgca 1080
          tattaccccg aagatttgtt tatcgacaag aaagtcttga aggtagccca tgttgaacac 1140
          gaagaaactc tctcctccag acgccgtgaa ctcattcaga aacttaagtc attcatatca 1200
          ttttactctg ctcttcccgg ctatatctgt tctcactcac cagtagccga gaatgatact 1260
          ctctgctgga acggtcagga gcttgttgag aggtactcac aaaaagctgc taggaacggc 1320
          atgaagaacc agtttaactt gcacgaattg aaaatgaaag gccctgagcc cgtagttagt 1380
          cagattatcg ataagttgaa gcacatcaac cagctgctca ggacaatgtc aatgcctaaa 1440
          gggcgcgtac ttgataagaa cttggacgag gagggttttg aatcaggtga ctgtggtgac 1500
          gatgaggacg agtgcatagg tggaagcgga gatggcatga taaaggttaa gaaccagctg 1560
          agatttctcg ctgaattggc ctatgatttg gatgtcgatg acgcccctgg aaatagtcaa 1620
          caggccaccc ctaaagacaa cgagatttcc acatttcaca atcttggcaa tgttcacagc 1680
          ccacttaaaa caatcaagcc ctgtcctcca tgcaaatgcc cagcacctaa cctcttgggt 1740
          ggaccatccg tcttcatctt ccctccaaag atcaaggatg tactcatgat ctccctgagc 1800
          cccatagtca catgtgtggt ggtggatgtg agcgaggatg acccagatgt ccagatcagc 1860
          tggtttgtga acaacgtgga agtacacaca gctcagacac aaacccatag agaggattac 1920
          aacagtactc tccgggtggt cagtgccctc cccatccagc accaggactg gatgagtggc 1980
          aaggagttca aatgcaaggt caacaacaaa gacctcccag cgcccatcga gagaaccatc 2040
          tcaaaaccca aagggtcagt aagagctcca caggtatatg tcttgcctcc accagaagaa 2100
          gagatgacta agaaacaggt cactctgacc tgcatggtca cagacttcat gcctgaagac 2160
          atttacgtgg agtggaccaa caacgggaaa acagagctaa actacaagaa cactgaacca 2220
          gtcctggact ctgatggttc ttacttcatg tacagcaagc tgagagtgga aaagaagaac 2280
          tgggtggaaa gaaatagcta ctcctgttca gtggtccacg agggtctgca caatcaccac 2340
          acgactaaga gcttctcccg gactccgggt aaatga 2376
           <![CDATA[ <210> 10]]>
           <![CDATA[ <211> 766]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 10]]>
          Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser
          1 5 10 15
          Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro
                      20 25 30
          Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys
                  35 40 45
          Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn
              50 55 60
          Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile
          65 70 75 80
          Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg
                          85 90 95
          His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser
                      100 105 110
          Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val
                  115 120 125
          Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn
              130 135 140
          Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn
          145 150 155 160
          Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly
                          165 170 175
          Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met
                      180 185 190
          Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala
                  195 200 205
          Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe
              210 215 220
          Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr
          225 230 235 240
          Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val
                          245 250 255
          Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp
                      260 265 270
          Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg
                  275 280 285
          Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His
              290 295 300
          Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr
          305 310 315 320
          Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala
                          325 330 335
          Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala
                      340 345 350
          His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile
                  355 360 365
          Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr
              370 375 380
          Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn
          385 390 395 400
          Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly
                          405 410 415
          Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu
                      420 425 430
          Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu
                  435 440 445
          Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu
              450 455 460
          Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu
          465 470 475 480
          Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu
                          485 490 495
          Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro
                      500 505 510
          Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe
                  515 520 525
          His Asn Leu Gly Asn Val His Ser Pro Leu Lys Ile Lys Pro Cys Pro
              530 535 540
          Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe
          545 550 555 560
          Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro
                          565 570 575
          Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val
                      580 585 590
          Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr
                  595 600 605
          Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala
              610 615 620
          Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
          625 630 635 640
          Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser
                          645 650 655
          Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro
                      660 665 670
          Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val
                  675 680 685
          Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly
              690 695 700
          Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp
          705 710 715 720
          Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp
                          725 730 735
          Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His
                      740 745 750
          Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
                  755 760 765
           <![CDATA[ <210> 11]]>
           <![CDATA[ <211> 2373]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: nucleotide sequence of mouse GPC3-mFc]]>
           <![CDATA[ <400> 11]]>
          atggctggta ctgttagaac cgcttgcctg ctcgttgcca tgctcctggg tcttggctgt 60
          cttggccaag cacagccacc accccacca gacgctacct gtcaccaagt gcgttcattc 120
          ttccaaaggc tccagcccgg tctcaagtgg gttcccgaaa cccctgtacc aggttccgac 180
          ctccaggttt gcttgccaaa agggcctaca tgttgttctc gcaagatgga agagaaatac 240
          caactcactg ccaggctcaa tatggagcaa ctgctccaat cagcatctat ggagcttaag 300
          ttcctcataa ttcaaaatgc agccgtattc caggaagcct tcgagatcgt cgtccgacac 360
          gcaaagaact atacaaatgc catgtttaag aacaactacc cttcactcac accccaagca 420
          ttcgaatttg taggagagtt ctttacagat gttagcttgt atattctcgg ttctgacata 480
          aatgttgacg acatggtaaa cgaactgttt gattctcttt ttccagtaat ctatacacaa 540
          atgatgaatc cagggttgcc agaaagtgtt ttggacataa atgagtgttt gcgcggggct 600
          agaagggatt tgaaggtttt tgggtcattt cccaagctca ttatgactca agttagtaaa 660
          tctttgcagg ttacaaggat cttccttcag gccttgaatc tggggatcga agtgattaac 720
          actaccgatc atctgaagtt tagcaaggac tgtggtcgaa tgctcactag aatgtggtat 780
          tgcagttatt gtcaaggtct catgatggtc aagccttgcg ggggttactg taacgtcgtg 840
          atgcaaggtt gtatggccgg tgtagttgaa atagataaat attggagaga gtacatcctc 900
          tctctcgaag aactggtcaa tggtatgtac cggatatatg acatggaaaa tgtgctcctc 960
          ggtctctttt ctacaatcca tgattctatt caatacgtgc aaaagaacgg aggtaagctt 1020
          acaactacta ttgggaaact gtgtgcccac agccagcaac gccagtatcg aagcgcttat 1080
          tatcccgaag acttgtttat agataagaag atccttaaag ttgcacacgt cgagcacgaa 1140
          gagactctct catctcgccg acgcgagctc attcaaaagt tgaagagctt tattaatttt 1200
          tactctgcac tgccagggta catctgttcc cacagcccag tagcagaaaa tgacaccctg 1260
          tgctggaatg gtcaagagct cgtggagagg tactctcaaa aagctgcacg gaacggcatg 1320
          aagaatcaat tcaatcttca tgaattgaag atgaaaggcc ctgaaccagt tgtatcccag 1380
          attatcgata agcttaagca tattaatcaa cttctccgta ctatgagcgt ccctaaaggt 1440
          aaagtactcg acaaaagcct tgatgaagaa ggactggaaa gtggcgattg tggagacgat 1500
          gaggacgagt gtattggatc atccggggac ggaatggtaa aggtaaaaaa tcaattgcgc 1560
          ttccttgcag aattggctta cgaccttgac gttgatgacg ctccaggcaa taaacagcac 1620
          gggaatcaga aggacaacga gataactaca tctcatagtg ttggcaacat gccctctcca 1680
          cttaaaacaa tcaagccctg tcctccatgc aaatgcccag cacctaacct cttgggtgga 1740
          ccatccgtct tcatcttccc tccaaagatc aaggatgtac tcatgatctc cctgagcccc 1800
          atagtcacat gtgtggtggt ggatgtgagc gaggatgacc cagatgtcca gatcagctgg 1860
          tttgtgaaca acgtggaagt acacacagct cagacacaaa cccatagaga ggattacaac 1920
          agtactctcc gggtggtcag tgccctcccc atccagcacc aggactggat gagtggcaag 1980
          gagttcaaat gcaaggtcaa caacaaagac ctcccagcgc ccatcgagag aaccatctca 2040
          aaacccaaag ggtcagtaag agctccacag gtatatgtct tgcctccacc agaagaagag 2100
          atgactaaga aacaggtcac tctgacctgc atggtcacag acttcatgcc tgaagacatt 2160
          tacgtggagt ggaccaacaa cgggaaaaca gagctaaact acaagaacac tgaaccagtc 2220
          ctggactctg atggttctta cttcatgtac agcaagctga gagtggaaaa gaagaactgg 2280
          gtggaaagaa atagctactc ctgttcagtg gtccacgagg gtctgcacaa tcaccacacg 2340
          actaagagct tctcccggac tccgggtaaa tga 2373
           <![CDATA[ <210> 12]]>
           <![CDATA[ <211> 766]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of mouse GPC3-mFc]]>
           <![CDATA[ <400> 12]]>
          Gln Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser Phe
          1 5 10 15
          Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro Val
                      20 25 30
          Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys Cys
                  35 40 45
          Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn Met
              50 55 60
          Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile Ile
          65 70 75 80
          Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg His
                          85 90 95
          Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser Leu
                      100 105 110
          Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val Ser
                  115 120 125
          Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn Glu
              130 135 140
          Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Met Met Asn Pro
          145 150 155 160
          Gly Leu Pro Glu Ser Val Leu Asp Ile Asn Glu Cys Leu Arg Gly Ala
                          165 170 175
          Arg Arg Asp Leu Lys Val Phe Gly Ser Phe Pro Lys Leu Ile Met Thr
                      180 185 190
          Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala Leu
                  195 200 205
          Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe Ser
              210 215 220
          Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr Cys
          225 230 235 240
          Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val Val
                          245 250 255
          Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp Arg
                      260 265 270
          Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg Ile
                  275 280 285
          Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His Asp
              290 295 300
          Ser Ile Gln Tyr Val Gln Lys Asn Gly Gly Lys Leu Thr Thr Thr Ile
          305 310 315 320
          Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala Tyr
                          325 330 335
          Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Ile Leu Lys Val Ala His
                      340 345 350
          Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile Gln
                  355 360 365
          Lys Leu Lys Ser Phe Ile Asn Phe Tyr Ser Ala Leu Pro Gly Tyr Ile
              370 375 380
          Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn Gly
          385 390 395 400
          Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly Met
                          405 410 415
          Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu Pro
                      420 425 430
          Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu Leu
                  435 440 445
          Arg Thr Met Ser Val Pro Lys Gly Lys Val Leu Asp Lys Ser Leu Asp
              450 455 460
          Glu Glu Gly Leu Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu Cys
          465 470 475 480
          Ile Gly Ser Ser Gly Asp Gly Met Val Lys Val Lys Asn Gln Leu Arg
                          485 490 495
          Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro Gly
                      500 505 510
          Asn Lys Gln His Gly Asn Gln Lys Asp Asn Glu Ile Thr Thr Ser His
                  515 520 525
          Ser Val Gly Asn Met Pro Ser Pro Leu Lys Thr Ile Lys Pro Cys Pro
              530 535 540
          Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe
          545 550 555 560
          Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro
                          565 570 575
          Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val
                      580 585 590
          Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr
                  595 600 605
          Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala
              610 615 620
          Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
          625 630 635 640
          Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser
                          645 650 655
          Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro
                      660 665 670
          Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val
                  675 680 685
          Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly
              690 695 700
          Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp
          705 710 715 720
          Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp
                          725 730 735
          Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His
                      740 745 750
          Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
                  755 760 765
           <![CDATA[ <210> 13]]>
           <![CDATA[ <211> 2361]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: nucleotide sequence of human GPC3-rFc]]>
           <![CDATA[ <400> 13]]>
          atggccggaa ctgtacggac agcatgtctc gtggtggcca tgctcttgag tctggacttt 60
          cccggccaag cccaacctcc ccctcctccc cccgatgcca cctgccatca ggttaggtca 120
          tttttccaaa ggcttcagcc cgggctgaag tgggttcctg aaacccctgt ccctgggtcc 180
          gatcttcagg tctgcctccc caaagggcct acctgctgtt cccgtaaaat ggaggagaag 240
          taccagttga ccgctagact caacatggag cagctgctcc agtcagcatc tatggagttg 300
          aagttcctga ttatacaaaa cgccgcagtt ttccaggagg catttgaaat tgtagtacgt 360
          cacgcaaaaa actacaccaa tgcaatgttc aagaataact atccctctct gacccctcaa 420
          gcctttgagt tcgtaggcga gttcttcacc gatgtgagtc tctatatcct cgggtcagat 480
          atcaacgtag atgacatggt aaatgaactt tttgacagcc tgtttcccgt tatttatacc 540
          cagctgatga accctggcct tccagactca gcactcgaca taaatgaatg cctgcggggg 600
          gccagacgcg acttgaaggt ttttgggaac tttccaaagc ttatcatgac acaagtttca 660
          aagtctttgc aagtgacccg tattttcctt caagctctca accttggcat tgaggtcata 720
          aataccaccg atcaccttaa gttcagcaaa gactgcggga gaatgctgac tcgcatgtgg 780
          tactgcagct attgccaggg gctgatgatg gtgaagcctt gtggaggcta ttgtaatgtg 840
          gtgatgcagg gatgtatggc aggcgtagta gagatcgaca aatattggcg tgagtacatc 900
          ctctctttgg aggaacttgt aaacggtatg taccgaatat atgacatgga aaacgtgctt 960
          ctgggactct tctcaaccat ccacgactca atccaatatg ttcaaaagaa cgcaggaaaa 1020
          cttactacaa ctattggaaa gctgtgtgca cattcacagc agcgtcagta taggagcgca 1080
          tattaccccg aagatttgtt tatcgacaag aaagtcttga aggtagccca tgttgaacac 1140
          gaagaaactc tctcctccag acgccgtgaa ctcattcaga aacttaagtc attcatatca 1200
          ttttactctg ctcttcccgg ctatatctgt tctcactcac cagtagccga gaatgatact 1260
          ctctgctgga acggtcagga gcttgttgag aggtactcac aaaaagctgc taggaacggc 1320
          atgaagaacc agtttaactt gcacgaattg aaaatgaaag gccctgagcc cgtagttagt 1380
          cagattatcg ataagttgaa gcacatcaac cagctgctca ggacaatgtc aatgcctaaa 1440
          gggcgcgtac ttgataagaa cttggacgag gagggttttg aatcaggtga ctgtggtgac 1500
          gatgaggacg agtgcatagg tggaagcgga gatggcatga taaaggttaa gaaccagctg 1560
          agatttctcg ctgaattggc ctatgatttg gatgtcgatg acgcccctgg aaatagtcaa 1620
          caggccaccc ctaaagacaa cgagatttcc acatttcaca atcttggcaa tgttcacagc 1680
          ccacttaaaa gcaagcccac gtgcccaccc cctgaactcc tggggggacc gtctgtcttc 1740
          atcttccccc caaaacccaa ggacaccctc atgatctcac gcacccccga ggtcacatgc 1800
          gtggtggtgg acgtgagcca ggatgacccc gaggtgcagt tcacatggta cataaacaac 1860
          gagcaggtgc gcaccgcccg gccgccgcta cgggagcagc agttcaacag cacgatccgc 1920
          gtggtcagca ccctccccat cgcgcaccag gactggctga ggggcaagga gttcaagtgc 1980
          aaagtccaca acaaggcact cccggccccc atcgagaaaa ccatctccaa agccagaggg 2040
          cagcccctgg agccgaaggt ctacaccatg ggccctcccc gggaggagct gagcagcagg 2100
          tcggtcagcc tgacctgcat gatcaacggc ttctaccctt ccgacatctc ggtggagtgg 2160
          gagaagaacg ggaaggcaga ggacaactac aagaccacgc cggccgtgct ggacagcgac 2220
          ggctcctact tcctctacag caagctctca gtgcccacga gtgagtggca gcggggcgac 2280
          gtcttcacct gctccgtgat gcacgaggcc ttgcacaacc actacacgca gaagtccatc 2340
          tcccgctctc cgggtaaatg a 2361
           <![CDATA[ <210> 14]]>
           <![CDATA[ <211> 762]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of human GPC3-rFc]]>
           <![CDATA[ <400> 14]]>
          Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser
          1 5 10 15
          Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro
                      20 25 30
          Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys
                  35 40 45
          Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn
              50 55 60
          Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile
          65 70 75 80
          Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg
                          85 90 95
          His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser
                      100 105 110
          Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val
                  115 120 125
          Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn
              130 135 140
          Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn
          145 150 155 160
          Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly
                          165 170 175
          Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met
                      180 185 190
          Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala
                  195 200 205
          Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe
              210 215 220
          Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr
          225 230 235 240
          Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val
                          245 250 255
          Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp
                      260 265 270
          Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg
                  275 280 285
          Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His
              290 295 300
          Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr
          305 310 315 320
          Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala
                          325 330 335
          Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala
                      340 345 350
          His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile
                  355 360 365
          Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr
              370 375 380
          Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn
          385 390 395 400
          Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly
                          405 410 415
          Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu
                      420 425 430
          Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu
                  435 440 445
          Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu
              450 455 460
          Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu
          465 470 475 480
          Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu
                          485 490 495
          Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro
                      500 505 510
          Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe
                  515 520 525
          His Asn Leu Gly Asn Val His Ser Pro Leu Lys Ser Lys Pro Thr Cys
              530 535 540
          Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
          545 550 555 560
          Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
                          565 570 575
          Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp
                      580 585 590
          Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu
                  595 600 605
          Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala
              610 615 620
          His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn
          625 630 635 640
          Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly
                          645 650 655
          Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu
                      660 665 670
          Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr
                  675 680 685
          Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp
              690 695 700
          Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe
          705 710 715 720
          Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp
                          725 730 735
          Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
                      740 745 750
          Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
                  755 760
           <![CDATA[ <210> 15]]>
           <![CDATA[ <211> 2358]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: nucleotide sequence of mouse GPC3-rFc]]>
           <![CDATA[ <400> 15]]>
          atggctggta ctgttagaac cgcttgcctg ctcgttgcca tgctcctggg tcttggctgt 60
          cttggccaag cacagccacc accccacca gacgctacct gtcaccaagt gcgttcattc 120
          ttccaaaggc tccagcccgg tctcaagtgg gttcccgaaa cccctgtacc aggttccgac 180
          ctccaggttt gcttgccaaa agggcctaca tgttgttctc gcaagatgga agagaaatac 240
          caactcactg ccaggctcaa tatggagcaa ctgctccaat cagcatctat ggagcttaag 300
          ttcctcataa ttcaaaatgc agccgtattc caggaagcct tcgagatcgt cgtccgacac 360
          gcaaagaact atacaaatgc catgtttaag aacaactacc cttcactcac accccaagca 420
          ttcgaatttg taggagagtt ctttacagat gttagcttgt atattctcgg ttctgacata 480
          aatgttgacg acatggtaaa cgaactgttt gattctcttt ttccagtaat ctatacacaa 540
          atgatgaatc cagggttgcc agaaagtgtt ttggacataa atgagtgttt gcgcggggct 600
          agaagggatt tgaaggtttt tgggtcattt cccaagctca ttatgactca agttagtaaa 660
          tctttgcagg ttacaaggat cttccttcag gccttgaatc tggggatcga agtgattaac 720
          actaccgatc atctgaagtt tagcaaggac tgtggtcgaa tgctcactag aatgtggtat 780
          tgcagttatt gtcaaggtct catgatggtc aagccttgcg ggggttactg taacgtcgtg 840
          atgcaaggtt gtatggccgg tgtagttgaa atagataaat attggagaga gtacatcctc 900
          tctctcgaag aactggtcaa tggtatgtac cggatatatg acatggaaaa tgtgctcctc 960
          ggtctctttt ctacaatcca tgattctatt caatacgtgc aaaagaacgg aggtaagctt 1020
          acaactacta ttgggaaact gtgtgcccac agccagcaac gccagtatcg aagcgcttat 1080
          tatcccgaag acttgtttat agataagaag atccttaaag ttgcacacgt cgagcacgaa 1140
          gagactctct catctcgccg acgcgagctc attcaaaagt tgaagagctt tattaatttt 1200
          tactctgcac tgccagggta catctgttcc cacagcccag tagcagaaaa tgacaccctg 1260
          tgctggaatg gtcaagagct cgtggagagg tactctcaaa aagctgcacg gaacggcatg 1320
          aagaatcaat tcaatcttca tgaattgaag atgaaaggcc ctgaaccagt tgtatcccag 1380
          attatcgata agcttaagca tattaatcaa cttctccgta ctatgagcgt ccctaaaggt 1440
          aaagtactcg acaaaagcct tgatgaagaa ggactggaaa gtggcgattg tggagacgat 1500
          gaggacgagt gtattggatc atccggggac ggaatggtaa aggtaaaaaa tcaattgcgc 1560
          ttccttgcag aattggctta cgaccttgac gttgatgacg ctccaggcaa taaacagcac 1620
          gggaatcaga aggacaacga gataactaca tctcatagtg ttggcaacat gccctctcca 1680
          cttaaaagca agcccacgtg cccaccccct gaactcctgg ggggaccgtc tgtcttcatc 1740
          ttccccccaa aacccaagga caccctcatg atctcacgca cccccgaggt cacatgcgtg 1800
          gtggtggacg tgagccagga tgaccccgag gtgcagttca catggtacat aaacaacgag 1860
          caggtgcgca ccgcccggcc gccgctacgg gagcagcagt tcaacagcac gatccgcgtg 1920
          gtcagcaccc tccccatcgc gcaccaggac tggctgaggg gcaaggagtt caagtgcaaa 1980
          gtccacaaca aggcactccc ggcccccatc gagaaaacca tctccaaagc cagagggcag 2040
          cccctggagc cgaaggtcta caccatgggc cctccccggg aggagctgag cagcaggtcg 2100
          gtcagcctga cctgcatgat caacggcttc tacccttccg acatctcggt ggagtgggag 2160
          aagaacggga aggcagagga caactacaag accacgccgg ccgtgctgga cagcgacggc 2220
          tcctacttcc tctacagcaa gctctcagtg cccacgagtg agtggcagcg gggcgacgtc 2280
          ttcacctgct ccgtgatgca cgaggccttg cacaaccact acacgcagaa gtccatctcc 2340
          cgctctccgg gtaaatga 2358
           <![CDATA[ <210> 16]]>
           <![CDATA[ <211> 761]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of mouse GPC3-rFc]]>
           <![CDATA[ <400> 16]]>
          Gln Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser Phe
          1 5 10 15
          Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro Val
                      20 25 30
          Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys Cys
                  35 40 45
          Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn Met
              50 55 60
          Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile Ile
          65 70 75 80
          Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg His
                          85 90 95
          Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser Leu
                      100 105 110
          Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val Ser
                  115 120 125
          Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn Glu
              130 135 140
          Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Met Met Asn Pro
          145 150 155 160
          Gly Leu Pro Glu Ser Val Leu Asp Ile Asn Glu Cys Leu Arg Gly Ala
                          165 170 175
          Arg Arg Asp Leu Lys Val Phe Gly Ser Phe Pro Lys Leu Ile Met Thr
                      180 185 190
          Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala Leu
                  195 200 205
          Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe Ser
              210 215 220
          Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr Cys
          225 230 235 240
          Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val Val
                          245 250 255
          Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp Arg
                      260 265 270
          Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg Ile
                  275 280 285
          Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His Asp
              290 295 300
          Ser Ile Gln Tyr Val Gln Lys Asn Gly Gly Lys Leu Thr Thr Thr Ile
          305 310 315 320
          Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala Tyr
                          325 330 335
          Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Ile Leu Lys Val Ala His
                      340 345 350
          Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile Gln
                  355 360 365
          Lys Leu Lys Ser Phe Ile Asn Phe Tyr Ser Ala Leu Pro Gly Tyr Ile
              370 375 380
          Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn Gly
          385 390 395 400
          Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly Met
                          405 410 415
          Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu Pro
                      420 425 430
          Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu Leu
                  435 440 445
          Arg Thr Met Ser Val Pro Lys Gly Lys Val Leu Asp Lys Ser Leu Asp
              450 455 460
          Glu Glu Gly Leu Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu Cys
          465 470 475 480
          Ile Gly Ser Ser Gly Asp Gly Met Val Lys Val Lys Asn Gln Leu Arg
                          485 490 495
          Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro Gly
                      500 505 510
          Asn Lys Gln His Gly Asn Gln Lys Asp Asn Glu Ile Thr Thr Ser His
                  515 520 525
          Ser Val Gly Asn Met Pro Ser Pro Leu Lys Ser Lys Pro Thr Cys Pro
              530 535 540
          Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
          545 550 555 560
          Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
                          565 570 575
          Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr
                      580 585 590
          Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln
                  595 600 605
          Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His
              610 615 620
          Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys
          625 630 635 640
          Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln
                          645 650 655
          Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu
                      660 665 670
          Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro
                  675 680 685
          Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn
              690 695 700
          Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu
          705 710 715 720
          Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val
                          725 730 735
          Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
                      740 745 750
          Lys Ser Ile Ser Arg Ser Pro Gly Lys
                  755 760
           <![CDATA[ <210> 17]]>
           <![CDATA[ <211> 539]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of soluble human GPC3]]>
           <![CDATA[ <400> 17]]>
          Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser
          1 5 10 15
          Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro
                      20 25 30
          Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys
                  35 40 45
          Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn
              50 55 60
          Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile
          65 70 75 80
          Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg
                          85 90 95
          His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser
                      100 105 110
          Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val
                  115 120 125
          Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn
              130 135 140
          Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn
          145 150 155 160
          Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly
                          165 170 175
          Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met
                      180 185 190
          Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala
                  195 200 205
          Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe
              210 215 220
          Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr
          225 230 235 240
          Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val
                          245 250 255
          Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp
                      260 265 270
          Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg
                  275 280 285
          Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His
              290 295 300
          Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr
          305 310 315 320
          Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala
                          325 330 335
          Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala
                      340 345 350
          His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile
                  355 360 365
          Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr
              370 375 380
          Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn
          385 390 395 400
          Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly
                          405 410 415
          Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu
                      420 425 430
          Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu
                  435 440 445
          Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu
              450 455 460
          Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu
          465 470 475 480
          Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu
                          485 490 495
          Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro
                      500 505 510
          Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe
                  515 520 525
          His Asn Leu Gly Asn Val His Ser Pro Leu Lys
              530 535
           <![CDATA[ <210> 18]]>
           <![CDATA[ <211> 767]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of human GPC3-GST]]>
           <![CDATA[ <400> 18]]>
          Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser
          1 5 10 15
          Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro
                      20 25 30
          Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys
                  35 40 45
          Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn
              50 55 60
          Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile
          65 70 75 80
          Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg
                          85 90 95
          His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser
                      100 105 110
          Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val
                  115 120 125
          Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn
              130 135 140
          Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn
          145 150 155 160
          Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly
                          165 170 175
          Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met
                      180 185 190
          Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala
                  195 200 205
          Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe
              210 215 220
          Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr
          225 230 235 240
          Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val
                          245 250 255
          Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp
                      260 265 270
          Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg
                  275 280 285
          Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His
              290 295 300
          Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr
          305 310 315 320
          Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala
                          325 330 335
          Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala
                      340 345 350
          His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile
                  355 360 365
          Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr
              370 375 380
          Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn
          385 390 395 400
          Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly
                          405 410 415
          Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu
                      420 425 430
          Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu
                  435 440 445
          Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu
              450 455 460
          Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu
          465 470 475 480
          Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu
                          485 490 495
          Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro
                      500 505 510
          Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe
                  515 520 525
          His Asn Leu Gly Asn Val His Ser Pro Leu Lys Leu Glu Val Leu Phe
              530 535 540
          Gln Gly Pro Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu
          545 550 555 560
          Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
                          565 570 575
          Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys
                      580 585 590
          Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly
                  595 600 605
          Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp
              610 615 620
          Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser
          625 630 635 640
          Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile
                          645 650 655
          Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys
                      660 665 670
          Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr
                  675 680 685
          Tyr Leu Asn Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp
              690 695 700
          Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe
          705 710 715 720
          Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
                          725 730 735
          Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly
                      740 745 750
          Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Lys Ser Asp
                  755 760 765
           <![CDATA[ <210> 19]]>
           <![CDATA[ <211> 2304]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: base sequence of human GPC3-GST]]>
           <![CDATA[ <400> 19]]>
          caacccccac ctcctccacc tgatgcaact tgtcatcaag tacgtagttt tttccagcgg 60
          ctgcagcccg gactgaagtg ggtgcccgag actcccgtac ctgggtcaga cctgcaggta 120
          tgtcttccta aaggccccac ttgttgcagc aggaaaatgg aggagaaata ccaactcacc 180
          gccagactta acatggaaca attgctgcag tccgcatcta tggagctcaa attcctgata 240
          attcaaaacg ctgcagtgtt ccaggaagca tttgaaatag tggttaggca tgcaaagaac 300
          tacacaaatg ccatgtttaa aaataactac ccctccctca caccacaagc cttcgaattt 360
          gttggagaat tcttcacaga cgtaagtctt tacatactgg gtagcgacat caacgtcgat 420
          gacatggtca atgaactctt cgactccctg ttccctgtga tctataccca actcatgaat 480
          ccaggtctgc ccgatagcgc cctcgacata aatgagtgcc ttcgcggagc ccgaagggat 540
          ctgaaagttt ttgggaattt tccaaagctc atcatgaccc aagtgagcaa gtctctgcag 600
          gtcacccgga ttttttctgca ggccctcaac ctgggcatag aagttataaa cacaacagat 660
          catctcaaat ttagcaaaga ttgcgggaga atgctgacac ggatgtggta ttgctcttat 720
          tgccagggac tgatgatggt taagccctgc ggaggttact gcaacgtagt gatgcaaggg 780
          tgcatggctg gagtggtgga gatagacaaa tactggaggg aatatatact tagtctggag 840
          gagttggtga acggtatgta taggatat gatatggaga acgttctgtt gggccttttc 900
          tcaactatcc atgactccat tcagtatgtt cagaagaacg ctggaaaact gaccacaacc 960
          attggtaagt tgtgtgccca ttctcagcag cggcaatatc gatccgctta ctaccccgag 1020
          gatctgttta tcgataagaa agttcttaag gtggctcatg tagagcatga agagactctg 1080
          tcaagccgga gacgcgagtt gatacagaag ctgaaatcat ttatttcctt ctactctgct 1140
          ctcccaggct acatctgttc tcatagtcct gttgcagaga acgataccct gtgctggaac 1200
          gggcaagagc ttgtagaacg ctatagccaa aaagcagctc gcaatggtat gaaaaaccag 1260
          ttcaatctgc atgaactgaa gatgaagggc cccgaacctg tagttagcca aatcattgat 1320
          aagttgaagc atataaatca acttctccga actatgtcca tgcccaaagg gcgcgtcctt 1380
          gacaaaaacc tggacgaaga ggggtttgag tccggcgact gcggtgatga cgaggacgaa 1440
          tgtatcggag ggtccggtga cggcatgata aaagtaaaga atcagctcag attcctcgca 1500
          gaacttgcat acgatcttga tgtagacgat gcccctggga attctcagca ggcaactcct 1560
          aaagataatg aaatttccac ttttcacaat ctcggaaatg tgcatagtcc cctgaaactt 1620
          gaggtattgt tccagggacc catgagccct atccttggat actggaaaat aaaaggactc 1680
          gtgcaaccca cccgcctgct gctggagtat ctggaagaaa agtacgagga gcatctttat 1740
          gaaagagacg aaggtgacaa gtggcgaaac aagaagtttg agcttggact tgagtttccc 1800
          aaccttccct attatatcga cggcgacgtt aaacttaccc aatcaatggc cattattaga 1860
          tatatagctg ataaacataa tatgctcggt ggatgtccca aagagcgcgc cgaaataagt 1920
          atgctggagg gcgctgtgct ggacattcgt tacggagtgt ccagaatcgc ttactcaaaa 1980
          gactttgaaa ccctcaaggt agacttcctc tcaaaattgc cagagatgtt gaagatgttt 2040
          gaagatcgac tctgtcataa aacttatctc aatggtgatc acgttacaca ccccgatttc 2100
          atgctttacg acgccttgga tgtcgtcttg tatatggatc ctatgtgtct tgatgctttc 2160
          cctaagttgg tatgcttcaa aaagagaatt gaagctattc cccagatcga caaatacctg 2220
          aaatccagca agtatattgc ctggcctctt caaggatggc aagccacatt cggaggtggc 2280
          gatcatcccc caaaatccga ttga 2304
           <![CDATA[ <210> 20]]>
           <![CDATA[ <211> 2412]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: base sequence of human GPC3-AA-hFc]]>
           <![CDATA[ <400> 20]]>
          atggctggca cagtgaggac tgcttgtctc gtagtcgcaa tgttgcttag cttggacttc 60
          ccaggtcaag cacaaccccc acccccaccc ccagatgcaa cttgtcacca ggttcggagc 120
          ttcttccagc gactgcagcc tggattgaag tgggttcctg agacaccagt tcctgggagt 180
          gatctccaag tctgtctccc taagggtccc acatgttgtt cacgaaagat ggaggaaaag 240
          tatcaattga cagcaagact gaacatggaa caacttctgc agtcagcatc aatggagctt 300
          aaattcctca ttatacaaaa tgctgctgtc ttccaggaag ccttcgaaat agtggttagg 360
          cacgccaaaa attacacaaa tgccatgttc aaaaacaact atccttccct caccccacag 420
          gcctttgaat ttgttggaga gtttttcact gatgttagtt tgtatatatt gggatcagac 480
          ataaatgtgg atgatatggt gaacgaattg tttgatagcc tgttccctgt catatacacc 540
          cagcttatga accccggttt gcctgacagt gcacttgaca ttaatgagtg tctgagaggt 600
          gctcgtaggg atctcaaggt gttcgggaac tttccaaagc ttataatgac tcaggtttca 660
          aagagtctgc aagtaactag gatctttttg caagccttga acttgggaat tgaggtaata 720
          aataccactg atcacctgaa attcagtaaa gattgtggga ggatgttgac tcgcatgtgg 780
          tattgcagtt attgtcaggg tttgatgatg gtcaaaccct gtggcggtta ttgcaacgta 840
          gtgatgcagg gctgcatggc tggagtagta gaaatagata agtactggcg cgagtacata 900
          ctgtcacttg aggagcttgt caatggaatg tataggatct atgacatgga aaatgttctc 960
          cttgggcttt tcagtacaat ccatgatagc atccaatacg ttcaaaagaa cgccgggaag 1020
          cttacaacca ccataggaaa attgtgcgct cattcacaac aacggcaata ccgcagcgca 1080
          tattatcctg aagacctctt tattgacaag aaggttctta aggtcgccca cgttgagcat 1140
          gaagaaacac tgagttcccg gcgccgcgag ctgatacaaa aattgaagtc tttcataagt 1200
          ttctactccg ccctgcccgg ttacatctgt tctcattccc ccgtagccga gaacgacact 1260
          ctctgctgga acggtcaaga gctggtcgag cggtacagtc aaaaggcagc caggaatgga 1320
          atgaaaaacc aatttaatct ccatgagttg aaaatgaagg gtcccgaacc agtggtcagt 1380
          cagataatag ataaacttaa acacataaat caacttctcc gaacaatgtc tatgcccaag 1440
          ggccgcgtac ttgacaagaa tttggacgag gaaggcttcg aggccgggga ttgcggcgac 1500
          gatgaggatg agtgtatcgg aggagctggg gatgggatga tcaaagttaa gaatcaactc 1560
          cgtttcctgg ctgagcttgc atacgacctt gatgtagacg acgcccccgg gaattcccag 1620
          caagctaccc caaaggacaa cgaaattagt acatttcata atctggggaa tgtgcactca 1680
          cctctgaaat ctagagcaga ctacaaggac gacgatgaca agactagtga caaaactcac 1740
          acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 1800
          ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 1860
          gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1920
          cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1980
          gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 2040
          aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 2100
          gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 2160
          ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 2220
          gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 2280
          ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 2340
          tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 2400
          ccgggtaaat ga 2412
           <![CDATA[ <210> 21]]>
           <![CDATA[ <211> 779]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of human GPC3-AA-hFc]]>
           <![CDATA[ <400> 21]]>
          Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser
          1 5 10 15
          Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro
                      20 25 30
          Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys
                  35 40 45
          Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn
              50 55 60
          Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile
          65 70 75 80
          Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg
                          85 90 95
          His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser
                      100 105 110
          Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val
                  115 120 125
          Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn
              130 135 140
          Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn
          145 150 155 160
          Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly
                          165 170 175
          Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met
                      180 185 190
          Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala
                  195 200 205
          Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe
              210 215 220
          Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr
          225 230 235 240
          Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val
                          245 250 255
          Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp
                      260 265 270
          Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg
                  275 280 285
          Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His
              290 295 300
          Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr
          305 310 315 320
          Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala
                          325 330 335
          Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala
                      340 345 350
          His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile
                  355 360 365
          Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr
              370 375 380
          Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn
          385 390 395 400
          Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly
                          405 410 415
          Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu
                      420 425 430
          Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu
                  435 440 445
          Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu
              450 455 460
          Asp Glu Glu Gly Phe Glu Ala Gly Asp Cys Gly Asp Asp Glu Asp Glu
          465 470 475 480
          Cys Ile Gly Gly Ala Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu
                          485 490 495
          Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro
                      500 505 510
          Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe
                  515 520 525
          His Asn Leu Gly Asn Val His Ser Pro Leu Lys Ser Arg Ala Asp Tyr
              530 535 540
          Lys Asp Asp Asp Asp Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro
          545 550 555 560
          Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
                          565 570 575
          Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
                      580 585 590
          Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
                  595 600 605
          Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
              610 615 620
          Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
          625 630 635 640
          Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
                          645 650 655
          Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
                      660 665 670
          Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
                  675 680 685
          Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
              690 695 700
          Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
          705 710 715 720
          Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
                          725 730 735
          Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
                      740 745 750
          Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
                  755 760 765
          Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
              770 775
           <![CDATA[ <210> 22]]>
           <![CDATA[ <211> 327]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: base sequence of A27]]>
           <![CDATA[ <400> 22]]>
          gaaatagtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60
          ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120
          cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180
          gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240
          cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctcc gtggacgttc 300
          ggccaaggga ccaaggtgga aatcaaa 327
           <![CDATA[ <210> 23]]>
           <![CDATA[ <211> 109]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A27]]>
           <![CDATA[ <400> 23]]>
          Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
          1 5 10 15
          Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
                      20 25 30
          Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
                  35 40 45
          Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
              50 55 60
          Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
          65 70 75 80
          Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro
                          85 90 95
          Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
                      100 105
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A27 CDR1]]>
           <![CDATA[ <400> 24]]>
          Arg Ala Ser Gln Ser Val Ser Ser Ser Ser Tyr Leu Ala
          1 5 10
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A27 CDR2]]>
           <![CDATA[ <400> 25]]>
          Gly Ala Ser Ser Arg Ala Thr
          1 5
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A27 CDR3]]>
           <![CDATA[ <400> 26]]>
          Gln Gln Tyr Gly Ser Ser Pro Pro Trp Thr
          1 5 10
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071 VH]]>
           <![CDATA[ <400> 27]]>
          Tyr 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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071 HCDR1]]>
           <![CDATA[ <400> 28]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071 HCDR2]]>
           <![CDATA[ <400> 29]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071 HCDR3]]>
           <![CDATA[ <400> 30]]>
          Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1007 VH]]>
           <![CDATA[ <400> 31]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Lys Gly Tyr
                      20 25 30
          Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ser Leu Ser Asn Gly Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Lys Thr Leu Gly Ala Tyr Tyr Ile Lys Ser Ala Met Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 32]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1007 HCDR1]]>
           <![CDATA[ <400> 32]]>
          Gly Tyr Ser Met Ser
          1 5
           <![CDATA[ <210> 33]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1007 HCDR2]]>
           <![CDATA[ <400> 33]]>
          Ser Leu Ser Asn Gly Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1007 HCDR3]]>
           <![CDATA[ <400> 34]]>
          Thr Leu Gly Ala Tyr Tyr Ile Lys Ser Ala Met Asp Val
          1 5 10
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071 VH starting from EVQL]]>
           <![CDATA[ <400> 35]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_201 VH]]>
           <![CDATA[ <400> 36]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Gly Ile Ser Asn Gly Ser Val Tyr Gln Trp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_201 HCDR1]]>
           <![CDATA[ <400> 37]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_201 HCDR2]]>
           <![CDATA[ <400> 38]]>
          Gly Ile Ser Asn Gly Ser Val Tyr Gln Trp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_201 HCDR3]]>
           <![CDATA[ <400> 39]]>
          Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_202 VH]]>
           <![CDATA[ <400> 40]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Trp Ile Ser Asn Gly Gly Val Phe Ser Gly Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 41]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_202 HCDR1]]>
           <![CDATA[ <400> 41]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 42]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_202 HCDR2]]>
           <![CDATA[ <400> 42]]>
          Trp Ile Ser Asn Gly Gly Val Phe Ser Gly Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 43]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_202 HCDR3]]>
           <![CDATA[ <400> 43]]>
          Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 44]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_203 VH]]>
           <![CDATA[ <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 Ser Phe Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Trp Ile Ser Asn Gly Gly Val Trp Lys Gly Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 45]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_203 HCDR1]]>
           <![CDATA[ <400> 45]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 46]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_203 HCDR2]]>
           <![CDATA[ <400> 46]]>
          Trp Ile Ser Asn Gly Gly Val Trp Lys Gly Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 47]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_203 HCDR3]]>
           <![CDATA[ <400> 47]]>
          Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 48]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_204 VH]]>
           <![CDATA[ <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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Trp Ile Ser Asn Gly Gly Val Phe Gly Gly Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 49]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_204 HCDR1]]>
           <![CDATA[ <400> 49]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 50]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_204 HCDR2]]>
           <![CDATA[ <400> 50]]>
          Trp Ile Ser Asn Gly Gly Val Phe Gly Gly Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 51]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_204 HCDR3]]>
           <![CDATA[ <400> 51]]>
          Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 52]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_301 VH]]>
           <![CDATA[ <400> 52]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Arg Ser Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 53]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_301 HCDR1]]>
           <![CDATA[ <400> 53]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 54]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_301 HCDR2]]>
           <![CDATA[ <400> 54]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 55]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_301 HCDR3]]>
           <![CDATA[ <400> 55]]>
          Ala Arg Ser Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 56]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_302 VH]]>
           <![CDATA[ <400> 56]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Arg Tyr Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 57]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_302 HCDR1]]>
           <![CDATA[ <400> 57]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 58]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_302 HCDR2]]>
           <![CDATA[ <400> 58]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 59]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_302 HCDR3]]>
           <![CDATA[ <400> 59]]>
          Ala Arg Tyr Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 60]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_303 VH]]>
           <![CDATA[ <400> 60]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Arg Ser Pro Trp Leu Tyr Arg Thr Gly Val Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 61]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_303 HCDR1]]>
           <![CDATA[ <400> 61]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 62]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_303 HCDR2]]>
           <![CDATA[ <400> 62]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 63]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_303 HCDR3]]>
           <![CDATA[ <400> 63]]>
          Ala Arg Ser Pro Trp Leu Tyr Arg Thr Gly Val Asp Val
          1 5 10
           <![CDATA[ <210> 64]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_304 VH]]>
           <![CDATA[ <400> 64]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Ala Glu Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 65]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_304 HCDR1]]>
           <![CDATA[ <400> 65]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 66]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_304 HCDR2]]>
           <![CDATA[ <400> 66]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 67]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_304 HCDR3]]>
           <![CDATA[ <400> 67]]>
          Ala Ser Gln Pro Trp Leu Tyr Ala Glu Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 68]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_305 VH]]>
           <![CDATA[ <400> 68]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Met Gly Val Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 69]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_305 HCDR1]]>
           <![CDATA[ <400> 69]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 70]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_305 HCDR2]]>
           <![CDATA[ <400> 70]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 71]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_305 HCDR3]]>
           <![CDATA[ <400> 71]]>
          Ala Ser Gln Pro Trp Leu Tyr Arg Met Gly Val Asp Val
          1 5 10
           <![CDATA[ <210> 72]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_306 VH]]>
           <![CDATA[ <400> 72]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Val Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 73]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_306 HCDR1]]>
           <![CDATA[ <400> 73]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 74]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_306 HCDR2]]>
           <![CDATA[ <400> 74]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 75]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_306 HCDR3]]>
           <![CDATA[ <400> 75]]>
          Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Val Asp Val
          1 5 10
           <![CDATA[ <210> 76]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR435 VH]]>
           <![CDATA[ <400> 76]]>
          Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Lys Ser Tyr
                      20 25 30
          Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Val Ile Ser Phe Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Asp Ser Asn Phe Trp Ser Gly Tyr Tyr Ser Pro Val Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 77]]>
           <![CDATA[ <211> 111]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR435 VL]]>
           <![CDATA[ <400> 77]]>
          Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys
          1 5 10 15
          Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn
                      20 25 30
          Ser Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Ile Thr Val
                  35 40 45
          Ile Tyr Glu Asp Thr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
              50 55 60
          Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly
          65 70 75 80
          Leu Gln Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser
                          85 90 95
          Ala Tyr His Trp Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu
                      100 105 110
           <![CDATA[ <210> 78]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98D VH]]>
           <![CDATA[ <400> 78]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Asp Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 79]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98D HCDR1]]>
           <![CDATA[ <400> 79]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 80]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98D HCDR2]]>
           <![CDATA[ <400> 80]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 81]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98D HCDR3]]>
           <![CDATA[ <400> 81]]>
          Ala Ser Gln Asp Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 82]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98E VH]]>
           <![CDATA[ <400> 82]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Glu Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 83]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98E HCDR1]]>
           <![CDATA[ <400> 83]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 84]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98E HCDR2]]>
           <![CDATA[ <400> 84]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 85]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98E HCDR3]]>
           <![CDATA[ <400> 85]]>
          Ala Ser Gln Glu Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 86]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98K VH]]>
           <![CDATA[ <400> 86]]>
          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 Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Lys Trp Leu Tyr Arg Thr Gly Ala Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 87]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98K HCDR1]]>
           <![CDATA[ <400> 87]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 88]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98K HCDR2]]>
           <![CDATA[ <400> 88]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 89]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_P98K HCDR3]]>
           <![CDATA[ <400> 89]]>
          Ala Ser Gln Lys Trp Leu Tyr Arg Thr Gly Ala Asp Val
          1 5 10
           <![CDATA[ <210> 90]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1015 VH]]>
           <![CDATA[ <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 Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Tyr Ile His Trp Val Gln Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Val Ile Asn Pro Ser Ile Val Ile Thr Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val 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 Glu Gly Gly Tyr Ser Gly Tyr Asp Pro Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 91]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1015 HCDR1]]>
           <![CDATA[ <400> 91]]>
          Ser Tyr Tyr Ile His
          1 5
           <![CDATA[ <210> 92]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1015 HCDR2]]>
           <![CDATA[ <400> 92]]>
          Val Ile Asn Pro Ser Ile Val Ile Thr Asn Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Asp
           <![CDATA[ <210> 93]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1015 HCDR3]]>
           <![CDATA[ <400> 93]]>
          Glu Gly Gly Tyr Ser Gly Tyr Asp Pro Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 94]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2002 VH]]>
           <![CDATA[ <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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Ala Val Ala Gly Gly His Tyr Tyr Tyr Gly Met
                      100 105 110
          Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 95]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2002 HCDR1]]>
           <![CDATA[ <400> 95]]>
          Ser Tyr Val Ile Gln
          1 5
           <![CDATA[ <210> 96]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2002 HCDR2]]>
           <![CDATA[ <400> 96]]>
          Trp Ile Asn Ala Gly Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 97]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2002 HCDR3]]>
           <![CDATA[ <400> 97]]>
          Gly Gly Ile Ala Val Ala Gly Gly His Tyr Tyr Tyr Gly Met Asp Ile
          1 5 10 15
           <![CDATA[ <210> 98]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1036 VH]]>
           <![CDATA[ <400> 98]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Ala Val Ala Gly Gly His Tyr Tyr Tyr Gly Met
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 99]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1036 HCDR1]]>
           <![CDATA[ <400> 99]]>
          Ser Tyr Val Ile Gln
          1 5
           <![CDATA[ <210> 100]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1036 HCDR2]]>
           <![CDATA[ <400> 100]]>
          Trp Ile Asn Ala Gly Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 101]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1036 HCDR3]]>
           <![CDATA[ <400> 101]]>
          Gly Gly Ile Ala Val Ala Gly Gly His Tyr Tyr Tyr Gly Met Asp Val
          1 5 10 15
           <![CDATA[ <210> 102]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2133 VH]]>
           <![CDATA[ <400> 102]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Met Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Ala Val Ala Gly Gly His Tyr Tyr Tyr Gly Met
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 103]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2133 HCDR1]]>
           <![CDATA[ <400> 103]]>
          Ser Tyr Val Met Gln
          1 5
           <![CDATA[ <210> 104]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2133 HCDR2]]>
           <![CDATA[ <400> 104]]>
          Trp Ile Asn Ala Gly Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 105]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2133 HCDR3]]>
           <![CDATA[ <400> 105]]>
          Gly Gly Ile Ala Val Ala Gly Gly His Tyr Tyr Tyr Gly Met Asp Val
          1 5 10 15
           <![CDATA[ <210> 106]]>
           <![CDATA[ <211> 126]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2067 VH]]>
           <![CDATA[ <400> 106]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Met Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Asp Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Asn 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 Gly Gly Ile Val Val Ala Gly Ser Gly Tyr Tyr Tyr Tyr Gly
                      100 105 110
          Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 107]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2067 HCDR1]]>
           <![CDATA[ <400> 107]]>
          Ser Tyr Val Leu His
          1 5
           <![CDATA[ <210> 108]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2067 HCDR2]]>
           <![CDATA[ <400> 108]]>
          Trp Ile Asn Ala Asp Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 109]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2067 HCDR3]]>
           <![CDATA[ <400> 109]]>
          Gly Gly Ile Val Val Ala Gly Ser Gly Tyr Tyr Tyr Tyr Gly Met Asp
          1 5 10 15
          Val
           <![CDATA[ <210> 110]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2103 VH]]>
           <![CDATA[ <400> 110]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Thr Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Val Val Ala Gly Gly His Tyr Tyr Tyr Gly Met
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 111]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2103 HCDR1]]>
           <![CDATA[ <400> 111]]>
          Ser Tyr Val Met His
          1 5
           <![CDATA[ <210> 112]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2103 HCDR2]]>
           <![CDATA[ <400> 112]]>
          Trp Ile Asn Ala Gly Thr Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 113]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2103 HCDR3]]>
           <![CDATA[ <400> 113]]>
          Gly Gly Ile Val Val Ala Gly Gly His Tyr Tyr Tyr Gly Met Asp Val
          1 5 10 15
           <![CDATA[ <210> 114]]>
           <![CDATA[ <211> 126]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1119 VH]]>
           <![CDATA[ <400> 114]]>
          Gln 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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Asp Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Val Val Ala Gly Ser Gly Tyr Tyr Tyr Tyr Gly
                      100 105 110
          Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 115]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1119 HCDR1]]>
           <![CDATA[ <400> 115]]>
          Ser Tyr Val Leu His
          1 5
           <![CDATA[ <210> 116]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1119 HCDR2]]>
           <![CDATA[ <400> 116]]>
          Trp Ile Asn Ala Asp Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 117]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1119 HCDR3]]>
           <![CDATA[ <400> 117]]>
          Gly Gly Ile Val Val Ala Gly Ser Gly Tyr Tyr Tyr Tyr Gly Met Asp
          1 5 10 15
          Val
           <![CDATA[ <210> 118]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3005 VH]]>
           <![CDATA[ <400> 118]]>
          Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Ile Ile Asn Pro Ser Val Gly Ser Thr Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
          65 70 75 80
          Met Glu Leu Ser Asn Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Glu Ser Gly Tyr Ser Gly Tyr Asp Ser Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 119]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3005 HCDR1]]>
           <![CDATA[ <400> 119]]>
          Ser Tyr Tyr Met His
          1 5
           <![CDATA[ <210> 120]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3005 HCDR2]]>
           <![CDATA[ <400> 120]]>
          Ile Ile Asn Pro Ser Val Gly Ser Thr Asn Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 121]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3005 HCDR3]]>
           <![CDATA[ <400> 121]]>
          Glu Ser Gly Tyr Ser Gly Tyr Asp Ser Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 122]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1016 VH]]>
           <![CDATA[ <400> 122]]>
          Glu Val Gln Leu Val Glu Ser Gly Thr Glu Val Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr
                      20 25 30
          Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Lys Leu Asn Pro Ile Gly Gly Asp Thr Ile Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Asn Arg Val Tyr
          65 70 75 80
          Met Glu Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Glu Gly Gly Tyr Ser Gly Tyr Asp Ala Phe Asp Ile Trp Gly
                      100 105 110
          Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 123]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1016 HCDR1]]>
           <![CDATA[ <400> 123]]>
          Arg Tyr Tyr Met His
          1 5
           <![CDATA[ <210> 124]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1016 HCDR2]]>
           <![CDATA[ <400> 124]]>
          Lys Leu Asn Pro Ile Gly Gly Asp Thr Ile Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 125]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1016 HCDR3]]>
           <![CDATA[ <400> 125]]>
          Glu Gly Gly Tyr Ser Gly Tyr Asp Ala Phe Asp Ile
          1 5 10
           <![CDATA[ <210> 126]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1054 VH]]>
           <![CDATA[ <400> 126]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          His Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Ile Ile Asn Pro Ser Leu Ser Asn Thr Asn Tyr Ala Leu Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Ile Arg Asp Thr Ser Thr Ser Thr Val Tyr
          65 70 75 80
          Met Glu Leu Thr Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Glu Gly Arg Tyr Ser Gly Tyr Asp Pro Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 127]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1054 HCDR1]]>
           <![CDATA[ <400> 127]]>
          Ser Tyr His Met His
          1 5
           <![CDATA[ <210> 128]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1054 HCDR2]]>
           <![CDATA[ <400> 128]]>
          Ile Ile Asn Pro Ser Leu Ser Asn Thr Asn Tyr Ala Leu Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 129]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1054 HCDR3]]>
           <![CDATA[ <400> 129]]>
          Glu Gly Arg Tyr Ser Gly Tyr Asp Pro Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 130]]>
           <![CDATA[ <211> 127]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2038 VH]]>
           <![CDATA[ <400> 130]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Val Ile Val Ser Pro Ala Gly Phe Tyr Tyr Tyr
                      100 105 110
          Gly Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 131]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2038 HCDR1]]>
           <![CDATA[ <400> 131]]>
          Ser Tyr Ala Met His
          1 5
           <![CDATA[ <210> 132]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2038 HCDR2]]>
           <![CDATA[ <400> 132]]>
          Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 133]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2038 HCDR3]]>
           <![CDATA[ <400> 133]]>
          Gly Gly Ile Val Ile Val Ser Pro Ala Gly Phe Tyr Tyr Tyr Gly Leu
          1 5 10 15
          Asp Val
           <![CDATA[ <210> 134]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1018 VH]]>
           <![CDATA[ <400> 134]]>
          Gln 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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Ala Val Ala Gly Gly Tyr Tyr Tyr Tyr Gly Met
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 135]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1018 HCDR1]]>
           <![CDATA[ <400> 135]]>
          Ser Tyr Ala Met His
          1 5
           <![CDATA[ <210> 136]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1018 HCDR2]]>
           <![CDATA[ <400> 136]]>
          Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 137]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1018 HCDR3]]>
           <![CDATA[ <400> 137]]>
          Gly Gly Ile Ala Val Ala Gly Gly Tyr Tyr Tyr Tyr Gly Met Asp Val
          1 5 10 15
           <![CDATA[ <210> 138]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2072 VH]]>
           <![CDATA[ <400> 138]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Ala Val Ala Gly Gly His Tyr Tyr Tyr Gly Met
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 139]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2072 HCDR1]]>
           <![CDATA[ <400> 139]]>
          Ser Tyr Val Met His
          1 5
           <![CDATA[ <210> 140]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2072 HCDR2]]>
           <![CDATA[ <400> 140]]>
          Trp Ile Asn Ala Gly Ser Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 141]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2072 HCDR3]]>
           <![CDATA[ <400> 141]]>
          Gly Gly Ile Ala Val Ala Gly Gly His Tyr Tyr Tyr Gly Met Asp Val
          1 5 10 15
           <![CDATA[ <210> 142]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4068 VH]]>
           <![CDATA[ <400> 142]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Arg Ala Ser Gly Gly Thr Phe Ser Thr Tyr
                      20 25 30
          Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Ile Pro Phe Phe Gly Ile Ala Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr 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 Lys Ile Tyr Tyr Asp Gly Ser Gly Ser Pro Leu Gly Met Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 143]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4068 HCDR1]]>
           <![CDATA[ <400> 143]]>
          Thr Tyr Ala Ile Ser
          1 5
           <![CDATA[ <210> 144]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4068 HCDR2]]>
           <![CDATA[ <400> 144]]>
          Gly Ile Ile Pro Phe Phe Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 145]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4068 HCDR3]]>
           <![CDATA[ <400> 145]]>
          Ile Tyr Tyr Asp Gly Ser Gly Ser Pro Leu Gly Met Asp Val
          1 5 10
           <![CDATA[ <210> 146]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4045 VH]]>
           <![CDATA[ <400> 146]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Asn Tyr
                      20 25 30
          Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Ile Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr 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 Asp Asn Tyr Tyr Gly Ser Gly Ser Pro Arg Gly Met Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 147]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4045 HCDR1]]>
           <![CDATA[ <400> 147]]>
          Asn Tyr Ala Ile Ser
          1 5
           <![CDATA[ <210> 148]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4045 HCDR2]]>
           <![CDATA[ <400> 148]]>
          Gly Ile Ile Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 149]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4045 HCDR3]]>
           <![CDATA[ <400> 149]]>
          Asp Asn Tyr Tyr Gly Ser Gly Ser Pro Arg Gly Met Asp Val
          1 5 10
           <![CDATA[ <210> 150]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1104 VH]]>
           <![CDATA[ <400> 150]]>
          Gln 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 Val Ser Gly His Thr Leu Thr Glu Leu
                      20 25 30
          Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp 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 Thr Glu Gly Leu Leu Trp Phe Gly Glu Phe Lys Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 151]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1104 HCDR1]]>
           <![CDATA[ <400> 151]]>
          Glu Leu Ser Ile His
          1 5
           <![CDATA[ <210> 152]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1104 HCDR2]]>
           <![CDATA[ <400> 152]]>
          Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 153]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1104 HCDR3]]>
           <![CDATA[ <400> 153]]>
          Glu Gly Leu Leu Trp Phe Gly Glu Phe Lys Tyr
          1 5 10
           <![CDATA[ <210> 154]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3117 VH]]>
           <![CDATA[ <400> 154]]>
          Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Gly Ala Ser Gly Phe Thr Phe Ser Thr Tyr
                      20 25 30
          Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Ala Ile Ser Ser Ser Gly Thr Ser Thr Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Leu Arg Asp Asn Ser Lys Asn Thr Leu Phe
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Lys Asp Arg Arg Gly Thr Met Gly Gly Phe Asp Tyr Trp Ala Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 155]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3117 HCDR1]]>
           <![CDATA[ <400> 155]]>
          Thr Tyr Ala Met Ser
          1 5
           <![CDATA[ <210> 156]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3117 HCDR2]]>
           <![CDATA[ <400> 156]]>
          Ala Ile Ser Ser Ser Gly Thr Ser Thr Tyr Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 157]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3117 HCDR3]]>
           <![CDATA[ <400> 157]]>
          Asp Arg Arg Gly Thr Met Gly Gly Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 158]]>
           <![CDATA[ <211> 125]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3024 VH]]>
           <![CDATA[ <400> 158]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr
                      20 25 30
          Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Ile Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Gly Gly Phe Gly Lys Leu Tyr Tyr Tyr Tyr Tyr Tyr Gly Met
                      100 105 110
          Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 159]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3024 HCDR1]]>
           <![CDATA[ <400> 159]]>
          Ser Tyr Trp Ile Gly
          1 5
           <![CDATA[ <210> 160]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3024 HCDR2]]>
           <![CDATA[ <400> 160]]>
          Ile Ile Tyr Pro Gly Asp Ser Asp Thr Ile Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 161]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3024 HCDR3]]>
           <![CDATA[ <400> 161]]>
          Gly Gly Phe Gly Lys Leu Tyr Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val
          1 5 10 15
           <![CDATA[ <210> 162]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3018 VH]]>
           <![CDATA[ <400> 162]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Thr Thr Asp
                      20 25 30
          Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Ile Pro Phe Phe Gly Ile Pro Asn Tyr Ala Arg Lys Phe
              50 55 60
          Gln Gly Ser Val Thr Ile Thr Ala Asp Ala Ser Thr Ser Thr Ala Tyr
          65 70 75 80
          Ile Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Thr Ser Tyr Tyr Gly Ser Gly Ser Pro Leu Gly Met Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 163]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3018 HCDR1]]>
           <![CDATA[ <400> 163]]>
          Thr Asp Ala Ile Asn
          1 5
           <![CDATA[ <210> 164]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3018 HCDR2]]>
           <![CDATA[ <400> 164]]>
          Gly Ile Ile Pro Phe Phe Gly Ile Pro Asn Tyr Ala Arg Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 165]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3018 HCDR3]]>
           <![CDATA[ <400> 165]]>
          Thr Ser Tyr Tyr Gly Ser Gly Ser Pro Leu Gly Met Asp Val
          1 5 10
           <![CDATA[ <210> 166]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3062 VH]]>
           <![CDATA[ <400> 166]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr
                      20 25 30
          Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Ile Pro Phe Phe Gly Ile Pro Asn Tyr Thr Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Ala Asp Asp Ser Thr 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 Glu Ala Tyr Tyr Gly Ser Gly Ile Pro His Gly Met Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 167]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3062 HCDR1]]>
           <![CDATA[ <400> 167]]>
          Asn Tyr Ala Ile Ser
          1 5
           <![CDATA[ <210> 168]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3062 HCDR2]]>
           <![CDATA[ <400> 168]]>
          Gly Ile Ile Pro Phe Phe Gly Ile Pro Asn Tyr Thr Gln Lys Phe Gln
          1 5 10 15
           <![CDATA[ <210> 169]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3062 HCDR3]]>
           <![CDATA[ <400> 169]]>
          Glu Ala Tyr Tyr Gly Ser Gly Ile Pro His Gly Met Asp Val
          1 5 10
           <![CDATA[ <210> 170]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3177 VH]]>
           <![CDATA[ <400> 170]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Tyr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile 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 Ile Leu Arg Tyr Phe Asp Trp Leu Tyr Ile Ala Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 171]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3177 HCDR1]]>
           <![CDATA[ <400> 171]]>
          Ser Tyr Tyr Ile Asn
          1 5
           <![CDATA[ <210> 172]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3177 HCDR2]]>
           <![CDATA[ <400> 172]]>
          Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln
          1 5 10 15
           <![CDATA[ <210> 173]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3177 HCDR3]]>
           <![CDATA[ <400> 173]]>
          Leu Arg Tyr Phe Asp Trp Leu Tyr Ile Ala Asp Tyr
          1 5 10
           <![CDATA[ <210> 174]]>
           <![CDATA[ <211> 126]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2024 VH]]>
           <![CDATA[ <400> 174]]>
          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 Thr Ser Gly Tyr Thr Phe Ser Ser Tyr
                      20 25 30
          Val Leu Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Asp Thr Asn Asn Thr Arg Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Ala Val Ala Gly Thr Gly Tyr Tyr Tyr Tyr Gly
                      100 105 110
          Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 175]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2024 HCDR1]]>
           <![CDATA[ <400> 175]]>
          Ser Tyr Val Leu Gln
          1 5
           <![CDATA[ <210> 176]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2024 HCDR2]]>
           <![CDATA[ <400> 176]]>
          Trp Ile Asn Ala Asp Thr Asn Asn Thr Arg Tyr Ser Gln Lys Phe Gln
          1 5 10 15
           <![CDATA[ <210> 177]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2024 HCDR3]]>
           <![CDATA[ <400> 177]]>
          Gly Gly Ile Ala Val Ala Gly Thr Gly Tyr Tyr Tyr Tyr Gly Met Asp
          1 5 10 15
          Val
           <![CDATA[ <210> 178]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1137 VH]]>
           <![CDATA[ <400> 178]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
                      20 25 30
          Ala Ile Ser Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Ile Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn 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 Glu Gly Tyr Tyr Gly Ser Gly Ser Pro Arg Gly Met Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 179]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1137 HCDR1]]>
           <![CDATA[ <400> 179]]>
          Ser Tyr Ala Ile Ser
          1 5
           <![CDATA[ <210> 180]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1137 HCDR2]]>
           <![CDATA[ <400> 180]]>
          Gly Ile Ile Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Asp
           <![CDATA[ <210> 181]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1137 HCDR3]]>
           <![CDATA[ <400> 181]]>
          Glu Gly Tyr Tyr Gly Ser Gly Ser Pro Arg Gly Met Asp Val
          1 5 10
           <![CDATA[ <210> 182]]>
           <![CDATA[ <211> 126]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2017 VH]]>
           <![CDATA[ <400> 182]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Thr Asn Asn Thr Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Ile Arg Asp Thr Ser Ala Ser Thr Ala Tyr
          65 70 75 80
          Val Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Gly Gly Ile Ala Val Ala Asp Thr Gly Tyr Tyr Tyr Tyr Gly
                      100 105 110
          Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 183]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2017 HCDR1]]>
           <![CDATA[ <400> 183]]>
          Ser Tyr Val Leu His
          1 5
           <![CDATA[ <210> 184]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2017 HCDR2]]>
           <![CDATA[ <400> 184]]>
          Trp Ile Asn Ala Gly Thr Asn Asn Thr Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
           <![CDATA[ <210> 185]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2017 HCDR3]]>
           <![CDATA[ <400> 185]]>
          Gly Gly Ile Ala Val Ala Asp Thr Gly Tyr Tyr Tyr Tyr Gly Met Asp
          1 5 10 15
          Val
           <![CDATA[ <210> 186]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1107 VH]]>
           <![CDATA[ <400> 186]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr
                      20 25 30
          Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Ile Pro Phe Phe Gly Ile Ala Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr 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 Ile His Tyr Asp Gly Ser Gly Ser Pro Leu Gly Met Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 187]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1107 HCDR1]]>
           <![CDATA[ <400> 187]]>
          Thr Tyr Ala Ile Ser
          1 5
           <![CDATA[ <210> 188]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1107 HCDR2]]>
           <![CDATA[ <400> 188]]>
          Gly Ile Ile Pro Phe Phe Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 189]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1107 HCDR3]]>
           <![CDATA[ <400> 189]]>
          Ile His Tyr Asp Gly Ser Gly Ser Pro Leu Gly Met Asp Val
          1 5 10
           <![CDATA[ <210> 190]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4025 VH]]>
           <![CDATA[ <400> 190]]>
          Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Asp Tyr
                      20 25 30
          Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Pro Val
                  35 40 45
          Ser Ala Ile Asn Thr Leu Gly Thr His Thr His Tyr Gly Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asp Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Ser Asp Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Val Ala Arg Val Pro Gly Phe Arg Pro Tyr Gln His Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 191]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4025 HCDR1]]>
           <![CDATA[ <400> 191]]>
          Asp Tyr Thr Met His
          1 5
           <![CDATA[ <210> 192]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4025 HCDR2]]>
           <![CDATA[ <400> 192]]>
          Ala Ile Asn Thr Leu Gly Thr His Thr His Tyr Gly Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 193]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4025 HCDR3]]>
           <![CDATA[ <400> 193]]>
          Arg Val Pro Gly Phe Arg Pro Tyr Gln His
          1 5 10
           <![CDATA[ <210> 194]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4009 VH]]>
           <![CDATA[ <400> 194]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Arg Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ala Ser Tyr
                      20 25 30
          Ala Met Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Thr Trp Ile
                  35 40 45
          Ser Ser Ile Asn Gly Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys
                          85 90 95
          Ala Lys His Gln Tyr Ser Val Tyr His Tyr Leu Gln Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 195]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4009 HCDR1]]>
           <![CDATA[ <400> 195]]>
          Ser Tyr Ala Met Ser
          1 5
           <![CDATA[ <210> 196]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4009 HCDR2]]>
           <![CDATA[ <400> 196]]>
          Ser Ile Asn Gly Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 197]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4009 HCDR3]]>
           <![CDATA[ <400> 197]]>
          His Gln Tyr Ser Val Tyr His Tyr Leu Gln Tyr
          1 5 10
           <![CDATA[ <210> 198]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3008 VH]]>
           <![CDATA[ <400> 198]]>
          Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Ile Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ser Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Lys Ile Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Thr Thr Asp Leu Ile Gly Val Ala Trp Gly Gln Gly Thr Leu
                      100 105 110
          Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 199]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3008 HCDR1]]>
           <![CDATA[ <400> 199]]>
          Asn Ala Trp Met Ser
          1 5
           <![CDATA[ <210> 200]]>
           <![CDATA[ <211> 19]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3008 HCDR2]]>
           <![CDATA[ <400> 200]]>
          Arg Ile Ile Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ser Ala Pro
          1 5 10 15
          Val Lys Gly
           <![CDATA[ <210> 201]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3008 HCDR3]]>
           <![CDATA[ <400> 201]]>
          Asp Leu Ile Gly Val Ala
          1 5
           <![CDATA[ <210> 202]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1048 VH]]>
           <![CDATA[ <400> 202]]>
          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 Val Ser Gly Tyr Thr Leu Thr Glu Leu
                      20 25 30
          Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp 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 Thr Glu Gly Leu Leu Trp Phe Gly Glu Phe Gly Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 203]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1048 HCDR1]]>
           <![CDATA[ <400> 203]]>
          Glu Leu Ser Ile His
          1 5
           <![CDATA[ <210> 204]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1048 HCDR2]]>
           <![CDATA[ <400> 204]]>
          Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 205]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1048 HCDR3]]>
           <![CDATA[ <400> 205]]>
          Glu Gly Leu Leu Trp Phe Gly Glu Phe Gly Tyr
          1 5 10
           <![CDATA[ <210> 206]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3042 VH]]>
           <![CDATA[ <400> 206]]>
          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 Val Ser Gly Tyr Thr Leu Thr Glu Leu
                      20 25 30
          Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Phe Asp Pro Glu Gly Gly Glu Thr Ile Tyr Ala Gln Asn Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp 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 Thr Glu Gly Leu Leu Trp Phe Gly Glu Leu Ser Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 207]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3042 HCDR1]]>
           <![CDATA[ <400> 207]]>
          Glu Leu Ser Ile His
          1 5
           <![CDATA[ <210> 208]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3042 HCDR2]]>
           <![CDATA[ <400> 208]]>
          Gly Phe Asp Pro Glu Gly Gly Glu Thr Ile Tyr Ala Gln Asn Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 209]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3042 HCDR3]]>
           <![CDATA[ <400> 209]]>
          Glu Gly Leu Leu Trp Phe Gly Glu Leu Ser Tyr
          1 5 10
           <![CDATA[ <210> 210]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3033 VH]]>
           <![CDATA[ <400> 210]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr
                      20 25 30
          Ala Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Ile Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
          65 70 75 80
          Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Ile His Tyr Ser Gly Ser Gly Ser Pro Leu Gly Met Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 211]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3033 HCDR1]]>
           <![CDATA[ <400> 211]]>
          Thr Tyr Ala Phe Ser
          1 5
           <![CDATA[ <210> 212]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3033 HCDR2]]>
           <![CDATA[ <400> 212]]>
          Gly Ile Ile Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 213]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3033 HCDR3]]>
           <![CDATA[ <400> 213]]>
          Ile His Tyr Ser Gly Ser Gly Ser Pro Leu Gly Met Asp Val
          1 5 10
           <![CDATA[ <210> 214]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4037 VH]]>
           <![CDATA[ <400> 214]]>
          Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Thr Thr Ser Gly Phe Thr Phe Arg Thr Tyr
                      20 25 30
          Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Gly Ser Gly Ala Val Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Thr
          65 70 75 80
          Leu Glu Met Asn Asn Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Asp Arg Tyr Gly Thr Pro Arg Arg Leu Phe Asp Gln Trp Gly
                      100 105 110
          Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 215]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4037 HCDR1]]>
           <![CDATA[ <400> 215]]>
          Thr Tyr Ala Met Ser
          1 5
           <![CDATA[ <210> 216]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4037 HCDR2]]>
           <![CDATA[ <400> 216]]>
          Val Ile Ser Gly Ser Gly Ala Val Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 217]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4037 HCDR3]]>
           <![CDATA[ <400> 217]]>
          Asp Arg Tyr Gly Thr Pro Arg Arg Leu Phe Asp Gln
          1 5 10
           <![CDATA[ <210> 218]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1122 VH]]>
           <![CDATA[ <400> 218]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Gln Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          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 Thr Thr Asp Leu Ile Ala Val Val Trp Gly Gln Gly Thr Leu
                      100 105 110
          Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 219]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1122 HCDR1]]>
           <![CDATA[ <400> 219]]>
          Asn Ala Trp Met Ser
          1 5
           <![CDATA[ <210> 220]]>
           <![CDATA[ <211> 19]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1122 HCDR2]]>
           <![CDATA[ <400> 220]]>
          Arg Ile Gln Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro
          1 5 10 15
          Val Lys Gly
           <![CDATA[ <210> 221]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G1122 HCDR3]]>
           <![CDATA[ <400> 221]]>
          Asp Leu Ile Ala Val Val
          1 5
           <![CDATA[ <210> 222]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2011 VH]]>
           <![CDATA[ <400> 222]]>
          Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          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 Thr Thr Asp Leu Ile Ala Val Ala Trp Gly Gln Gly Thr Thr
                      100 105 110
          Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 223]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2011 HCDR1]]>
           <![CDATA[ <400> 223]]>
          Asn Ala Trp Met Ser
          1 5
           <![CDATA[ <210> 224]]>
           <![CDATA[ <211> 19]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2011 HCDR2]]>
           <![CDATA[ <400> 224]]>
          Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro
          1 5 10 15
          Val Lys Gly
           <![CDATA[ <210> 225]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2011 HCDR3]]>
           <![CDATA[ <400> 225]]>
          Asp Leu Ile Ala Val Ala
          1 5
           <![CDATA[ <210> 226]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2025 VH]]>
           <![CDATA[ <400> 226]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Lys Gly Ser Gly Tyr Ser Phe Pro Ser Tyr
                      20 25 30
          Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Leu Ser Gln Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
                      100 105 110
          Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 227]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2025 HCDR1]]>
           <![CDATA[ <400> 227]]>
          Ser Tyr Trp Ile Gly
          1 5
           <![CDATA[ <210> 228]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2025 HCDR2]]>
           <![CDATA[ <400> 228]]>
          Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 229]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2025 HCDR3]]>
           <![CDATA[ <400> 229]]>
          Ser Gln Gly Asp Tyr Phe Asp Tyr
          1 5
           <![CDATA[ <210> 230]]>
           <![CDATA[ <211> 115]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4033 VH]]>
           <![CDATA[ <400> 230]]>
          Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Thr Tyr
                      20 25 30
          Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Asp Tyr Gly Asp Ser Val
              50 55 60
          Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Met Leu Tyr
          65 70 75 80
          Leu Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Asp Leu Phe Ala Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
                      100 105 110
          Val Ser Ser
                  115
           <![CDATA[ <210> 231]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4033 HCDR1]]>
           <![CDATA[ <400> 231]]>
          Thr Tyr Ala Met His
          1 5
           <![CDATA[ <210> 232]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4033 HCDR2]]>
           <![CDATA[ <400> 232]]>
          Val Ile Ser Tyr Asp Gly Ser Asn Lys Asp Tyr Gly Asp Ser Val Lys
          1 5 10 15
          Asp
           <![CDATA[ <210> 233]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G4033 HCDR3]]>
           <![CDATA[ <400> 233]]>
          Asp Leu Phe Ala Asp Tyr
          1 5
           <![CDATA[ <210> 234]]>
           <![CDATA[ <211> 123]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3062_NYA VH]]>
           <![CDATA[ <400> 234]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys Pro Gly Ser
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr
                      20 25 30
          Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Gly Ile Ile Pro Phe Phe Gly Ile Pro Asn Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Ala Asp Asp Ser Thr 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 Glu Ala Tyr Tyr Gly Ser Gly Ile Pro His Gly Met Asp Val
                      100 105 110
          Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 235]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3062_NYA HCDR1]]>
           <![CDATA[ <400> 235]]>
          Asn Tyr Ala Ile Ser
          1 5
           <![CDATA[ <210> 236]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3062_NYA HCDR2]]>
           <![CDATA[ <400> 236]]>
          Gly Ile Ile Pro Phe Phe Gly Ile Pro Asn Tyr Ala Gln Lys Phe Gln
          1 5 10 15
           <![CDATA[ <210> 237]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3062_NYA HCDR3]]>
           <![CDATA[ <400> 237]]>
          Glu Ala Tyr Tyr Gly Ser Gly Ile Pro His Gly Met Asp Val
          1 5 10
           <![CDATA[ <210> 238]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3177_NTA VH]]>
           <![CDATA[ <400> 238]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Tyr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ala Ile 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 Ile Leu Arg Tyr Phe Asp Trp Leu Tyr Ile Ala Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 239]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3177_NTA HCDR1]]>
           <![CDATA[ <400> 239]]>
          Ser Tyr Tyr Ile Asn
          1 5
           <![CDATA[ <210> 240]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3177_NTA HCDR2]]>
           <![CDATA[ <400> 240]]>
          Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln
          1 5 10 15
           <![CDATA[ <210> 241]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G3177_NTA HCDR3]]>
           <![CDATA[ <400> 241]]>
          Leu Arg Tyr Phe Asp Trp Leu Tyr Ile Ala Asp Tyr
          1 5 10
           <![CDATA[ <210> 242]]>
           <![CDATA[ <211> 126]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2024_NNA VH]]>
           <![CDATA[ <400> 242]]>
          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 Thr Ser Gly Tyr Thr Phe Ser Ser Tyr
                      20 25 30
          Val Leu Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Asp Thr Asn Asn Ala Arg Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Thr Arg 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 Gly Gly Ile Ala Val Ala Gly Thr Gly Tyr Tyr Tyr Tyr Gly
                      100 105 110
          Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 243]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2024_NNA HCDR1]]>
           <![CDATA[ <400> 243]]>
          Ser Tyr Val Leu Gln
          1 5
           <![CDATA[ <210> 244]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2024_NNA HCDR2]]>
           <![CDATA[ <400> 244]]>
          Trp Ile Asn Ala Asp Thr Asn Asn Ala Arg Tyr Ser Gln Lys Phe Gln
          1 5 10 15
           <![CDATA[ <210> 245]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2024_NNA HCDR3]]>
           <![CDATA[ <400> 245]]>
          Gly Gly Ile Ala Val Ala Gly Thr Gly Tyr Tyr Tyr Tyr Gly Met Asp
          1 5 10 15
          Val
           <![CDATA[ <210> 246]]>
           <![CDATA[ <211> 126]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2017_NNA VH]]>
           <![CDATA[ <400> 246]]>
          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 Tyr Thr Phe Thr Ser Tyr
                      20 25 30
          Val Leu His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Ala Gly Thr Asn Asn Ala Lys Tyr Ser Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Ile Ile Arg Asp Thr Ser Ala Ser Thr Ala Tyr
          65 70 75 80
          Val Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Ala Arg Gly Gly Ile Ala Val Ala Asp Thr Gly Tyr Tyr Tyr Tyr Gly
                      100 105 110
          Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
                  115 120 125
           <![CDATA[ <210> 247]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2017_NNA HCDR1]]>
           <![CDATA[ <400> 247]]>
          Ser Tyr Val Leu His
          1 5
           <![CDATA[ <210> 248]]>
           <![CDATA[ <211> 16]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2017_NNA HCDR2]]>
           <![CDATA[ <400> 248]]>
          Trp Ile Asn Ala Gly Thr Asn Asn Ala Lys Tyr Ser Gln Lys Phe Gln
          1 5 10 15
           <![CDATA[ <210> 249]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of G2017_NNA HCDR3]]>
           <![CDATA[ <400> 249]]>
          Gly Gly Ile Ala Val Ala Asp Thr Gly Tyr Tyr Tyr Tyr Gly Met Asp
          1 5 10 15
          Val
           <![CDATA[ <210> 250]]>
           <![CDATA[ <211> 117]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of HN3 VH]]>
           <![CDATA[ <400> 250]]>
          Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe Asp Ser Tyr
                      20 25 30
          Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
                  35 40 45
          Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
              50 55 60
          Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
          65 70 75 80
          Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
                          85 90 95
          Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
                      100 105 110
          Thr Val Ser Ser Ser
                  115
           <![CDATA[ <210> 251]]>
           <![CDATA[ <211> 229]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of Fc of IgG4PE R409K]]>
           <![CDATA[ <400> 251]]>
          Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
          1 5 10 15
          Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
                      20 25 30
          Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
                  35 40 45
          Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
              50 55 60
          Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
          65 70 75 80
          Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
                          85 90 95
          Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
                      100 105 110
          Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
                  115 120 125
          Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
              130 135 140
          Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
          145 150 155 160
          Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
                          165 170 175
          Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
                      180 185 190
          Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
                  195 200 205
          Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
              210 215 220
          Leu Ser Leu Gly Lys
          225
           <![CDATA[ <210> 252]]>
           <![CDATA[ <211> 294]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: base sequence of IgG4 CH1]]>
           <![CDATA[ <400> 252]]>
          gctagtacca aggggccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60
          agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
          tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
          ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240
          tacacctgca acgtagatca caagcccagc aacaccaagg tggataagag agtt 294
           <![CDATA[ <210> 253]]>
           <![CDATA[ <211> 98]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of Artificial Sequences: Amino Acid Sequences of Synthetic Structures]]>
           <![CDATA[ <400> 253]]>
          Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
          1 5 10 15
          Ser Thr Ser Glu Ser 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 Ser Leu Gly Thr Lys Thr
          65 70 75 80
          Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
                          85 90 95
          Arg Val
           <![CDATA[ <210> 254]]>
           <![CDATA[ <211> 981]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: base sequence of IgG4PE R409K CH]]>
           <![CDATA[ <400> 254]]>
          gctagcacca aggggccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60
          agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
          tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
          ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240
          tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300
          aaatatggtc ccccatgccc accatgccca gcacctgagt tcgagggggg accatcagtc 360
          ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420
          tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480
          ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540
          cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600
          tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660
          gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720
          aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780
          tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840
          gacggctcct tcttcctcta cagcaagcta accgtggaca agagcaggtg gcaggagggg 900
          aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960
          ctctccctgt ctctgggtaa a 981
           <![CDATA[ <210> 255]]>
           <![CDATA[ <211> 327]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of Artificial Sequences: Amino Acid Sequences of Synthetic Structures]]>
           <![CDATA[ <400> 255]]>
          Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
          1 5 10 15
          Ser Thr Ser Glu Ser 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 Ser Leu Gly Thr Lys Thr
          65 70 75 80
          Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
                          85 90 95
          Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
                      100 105 110
          Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
                  115 120 125
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
              130 135 140
          Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
          145 150 155 160
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
                          165 170 175
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
                      180 185 190
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
                  195 200 205
          Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
              210 215 220
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
          225 230 235 240
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
                          245 250 255
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
                      260 265 270
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
                  275 280 285
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
              290 295 300
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
          305 310 315 320
          Leu Ser Leu Ser Leu Gly Lys
                          325
           <![CDATA[ <210> 256]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of linker]]>
           <![CDATA[ <400> 256]]>
          Gly Gly Gly Gly Ser
          1 5
           <![CDATA[ <210> 257]]>
           <![CDATA[ <211> 366]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: base sequence of TfR1071 VH starting from EVQL]]>
           <![CDATA[ <400> 257]]>
          gaagtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
          tcctgtgcag cctctggatt cagctttgac aaatatacca tgaactgggt ccgccaggct 120
          ccagggaagg ggctggagtg ggtctcagtt atttctaatg ggggagtttc tacagactac 180
          gcagactccg ttaagggccg gttcaccgtc tccagagaca attccaagaa cacactgtac 240
          ctgcaaatga acagcctgag agccgaggat atggccacat attactgtgc gcgtgcgagc 300
          cagccgtggc tctataggac cggtgcggat gtgtggggcc aggggaccct ggtcaccgtc 360
          tcctca 366
           <![CDATA[ <210> 258]]>
           <![CDATA[ <211> 297]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: base sequence of IgG4 CH1 including stop codon]]>
           <![CDATA[ <400> 258]]>
          gctagcacca aaggaccttc tgtatttcct cttgcgccat gctctcgctc tacgtcagaa 60
          tcaactgccg ctctggggtg cctggttaaa gactacttcc cggagcctgt gacagtgagt 120
          tggaactccg gcgccctgac atcaggagtg catacatttc ccgccgtgct tcagagcagc 180
          ggactttata gcctcagcag tgtggtgacc gtgccatctt ccagcctggg gaccaagacc 240
          tacacctgta acgtggacca caaacccagc aacaccaagg ttgataagag ggtctga 297
           <![CDATA[ <210> 259]]>
           <![CDATA[ <211> 763]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequences: amino acid sequence of mouse TfR]]>
           <![CDATA[ <400> 259]]>
          Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
          1 5 10 15
          Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
                      20 25 30
          Asn Ser His Val Glu Met Lys Leu Ala Ala Asp Glu Glu Glu Asn Ala
                  35 40 45
          Asp Asn Asn Met Lys Ala Ser Val Arg Lys Pro Lys Arg Phe Asn Gly
              50 55 60
          Arg Leu Cys Phe Ala Ala Ile Ala Leu Val Ile Phe Phe Leu Ile Gly
          65 70 75 80
          Phe Met Ser Gly Tyr Leu Gly Tyr Cys Lys Arg Val Glu Gln Lys Glu
                          85 90 95
          Glu Cys Val Lys Leu Ala Glu Thr Glu Glu Thr Asp Lys Ser Glu Thr
                      100 105 110
          Met Glu Thr Glu Asp Val Pro Thr Ser Ser Arg Leu Tyr Trp Ala Asp
                  115 120 125
          Leu Lys Thr Leu Leu Ser Glu Lys Leu Asn Ser Ile Glu Phe Ala Asp
              130 135 140
          Thr Ile Lys Gln Leu Ser Gln Asn Thr Tyr Thr Pro Arg Glu Ala Gly
          145 150 155 160
          Ser Gln Lys Asp Glu Ser Leu Ala Tyr Tyr Ile Glu Asn Gln Phe His
                          165 170 175
          Glu Phe Lys Phe Ser Lys Val Trp Arg Asp Glu His Tyr Val Lys Ile
                      180 185 190
          Gln Val Lys Ser Ser Ile Gly Gln Asn Met Val Thr Ile Val Gln Ser
                  195 200 205
          Asn Gly Asn Leu Asp Pro Val Glu Ser Pro Glu Gly Tyr Val Ala Phe
              210 215 220
          Ser Lys Pro Thr Glu Val Ser Gly Lys Leu Val His Ala Asn Phe Gly
          225 230 235 240
          Thr Lys Lys Asp Phe Glu Glu Leu Ser Tyr Ser Val Asn Gly Ser Leu
                          245 250 255
          Val Ile Val Arg Ala Gly Glu Ile Thr Phe Ala Glu Lys Val Ala Asn
                      260 265 270
          Ala Gln Ser Phe Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Lys Asn
                  275 280 285
          Lys Phe Pro Val Val Glu Ala Asp Leu Ala Leu Phe Gly His Ala His
              290 295 300
          Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His
          305 310 315 320
          Thr Gln Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val
                          325 330 335
          Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Lys Met Glu
                      340 345 350
          Gly Ser Cys Pro Ala Arg Trp Asn Ile Asp Ser Ser Cys Lys Leu Glu
                  355 360 365
          Leu Ser Gln Asn Gln Asn Val Lys Leu Ile Val Lys Asn Val Leu Lys
              370 375 380
          Glu Arg Arg Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Tyr Glu Glu
          385 390 395 400
          Pro Asp Arg Tyr Val Val Val Gly Ala Gln Arg Asp Ala Leu Gly Ala
                          405 410 415
          Gly Val Ala Ala Lys Ser Ser Val Gly Thr Gly Leu Leu Leu Lys Leu
                      420 425 430
          Ala Gln Val Phe Ser Asp Met Ile Ser Lys Asp Gly Phe Arg Pro Ser
                  435 440 445
          Arg Ser Ile Ile Phe Ala Ser Trp Thr Ala Gly Asp Phe Gly Ala Val
              450 455 460
          Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys
          465 470 475 480
          Ala Phe Thr Tyr Ile Asn Leu Asp Lys Val Val Leu Gly Thr Ser Asn
                          485 490 495
          Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr Leu Met Gly Lys Ile
                      500 505 510
          Met Gln Asp Val Lys His Pro Val Asp Gly Lys Ser Leu Tyr Arg Asp
                  515 520 525
          Ser Asn Trp Ile Ser Lys Val Glu Lys Leu Ser Phe Asp Asn Ala Ala
              530 535 540
          Tyr Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe
          545 550 555 560
          Cys Glu Asp Ala Asp Tyr Pro Tyr Leu Gly Thr Arg Leu Asp Thr Tyr
                          565 570 575
          Glu Ala Leu Thr Gln Lys Val Pro Gln Leu Asn Gln Met Val Arg Thr
                      580 585 590
          Ala Ala Glu Val Ala Gly Gln Leu Ile Ile Lys Leu Thr His Asp Val
                  595 600 605
          Glu Leu Asn Leu Asp Tyr Glu Met Tyr Asn Ser Lys Leu Leu Ser Phe
              610 615 620
          Met Lys Asp Leu Asn Gln Phe Lys Thr Asp Ile Arg Asp Met Gly Leu
          625 630 635 640
          Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp Tyr Phe Arg Ala Thr
                          645 650 655
          Ser Arg Leu Thr Thr Asp Phe His Asn Ala Glu Lys Thr Asn Arg Phe
                      660 665 670
          Val Met Arg Glu Ile Asn Asp Arg Ile Met Lys Val Glu Tyr His Phe
                  675 680 685
          Leu Ser Pro Tyr Val Ser Pro Arg Glu Ser Pro Phe Arg His Ile Phe
              690 695 700
          Trp Gly Ser Gly Ser His Thr Leu Ser Ala Leu Val Glu Asn Leu Lys
          705 710 715 720
          Leu Arg Gln Lys Asn Ile Thr Ala Phe Asn Glu Thr Leu Phe Arg Asn
                          725 730 735
          Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Val Ala Asn Ala Leu
                      740 745 750
          Ser Gly Asp Ile Trp Asn Ile Asp Asn Glu Phe
                  755 760
           <![CDATA[ <210> 260]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of mouse chimera of apical domain of human TfR extracellular domain]]>
           <![CDATA[ <400> 260]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Ser Ser Ile Gly Gln Asn Met
                      100 105 110
          Val Thr Ile Val Gln Ser Asn Gly Asn Leu Asp Pro Val Glu Ser Pro
                  115 120 125
          Glu Gly Tyr Val Ala Phe Ser Lys Pro Thr Glu Val Ser Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Glu Leu Ser Tyr
          145 150 155 160
          Ser Val Asn Gly Ser Leu Val Ile Val Arg Ala Gly Glu Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Gln Ser Phe Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Lys Asn Lys Phe Pro Val Val Glu Ala Asp Leu Ala
                  195 200 205
          Leu Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Gln Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Lys Met Glu Gly Ser Cys Pro Ala Arg Trp Asn Ile Asp
                      260 265 270
          Ser Ser Cys Lys Leu Glu Leu Ser Gln Asn Gln Asn Val Lys Leu Ile
                  275 280 285
          Val Lys Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
                      420 425 430
          Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
                          565 570 575
          Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
              610 615 620
          Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 261]]>
           <![CDATA[ <211> 675]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of Artificial Sequences: Amino Acid Sequence of Mouse Chimera of Protease-Like Domain of Human TfR Extracellular Region]]>
           <![CDATA[ <400> 261]]>
          Cys Lys Arg Val Glu Gln Lys Glu Glu Cys Val Lys Leu Ala Glu Thr
          1 5 10 15
          Glu Glu Thr Asp Lys Ser Glu Thr Met Glu Thr Glu Asp Val Pro Thr
                      20 25 30
          Ser Ser Arg Leu Tyr Trp Ala Asp Leu Lys Thr Leu Leu Ser Glu Lys
                  35 40 45
          Leu Asn Ser Ile Glu Phe Ala Asp Thr Ile Lys Gln Leu Ser Gln Asn
              50 55 60
          Thr Tyr Thr Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Ser Leu Ala
          65 70 75 80
          Tyr Tyr Ile Glu Asn Gln Phe His Glu Phe Lys Phe Ser Lys Val Trp
                          85 90 95
          Arg Asp Glu His Tyr Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn
                      100 105 110
          Ser Val Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu
                  115 120 125
          Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly
              130 135 140
          Lys Leu Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu
          145 150 155 160
          Tyr Thr Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile
                          165 170 175
          Thr Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly
                      180 185 190
          Val Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu
                  195 200 205
          Leu Ser Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr
              210 215 220
          Pro Gly Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser
          225 230 235 240
          Ser Gly Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala
                          245 250 255
          Glu Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys
                      260 265 270
          Thr Asp Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys
                  275 280 285
          Leu Thr Val Ser Asn Val Leu Lys Glu Arg Arg Ile Leu Asn Ile Phe
              290 295 300
          Gly Val Ile Lys Gly Tyr Glu Glu Pro Asp Arg Tyr Val Val Val Gly
          305 310 315 320
          Ala Gln Arg Asp Ala Leu Gly Ala Gly Val Ala Ala Lys Ser Ser Val
                          325 330 335
          Gly Thr Gly Leu Leu Leu Lys Leu Ala Gln Val Phe Ser Asp Met Ile
                      340 345 350
          Ser Lys Asp Gly Phe Arg Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp
                  355 360 365
          Thr Ala Gly Asp Phe Gly Ala Val Gly Ala Thr Glu Trp Leu Glu Gly
              370 375 380
          Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp
          385 390 395 400
          Lys Val Val Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu
                          405 410 415
          Leu Tyr Thr Leu Met Gly Lys Ile Met Gln Asp Val Lys His Pro Val
                      420 425 430
          Asp Gly Lys Ser Leu Tyr Arg Asp Ser Asn Trp Ile Ser Lys Val Glu
                  435 440 445
          Lys Leu Ser Phe Asp Asn Ala Ala Tyr Pro Phe Leu Ala Tyr Ser Gly
              450 455 460
          Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp Ala Asp Tyr Pro Tyr
          465 470 475 480
          Leu Gly Thr Arg Leu Asp Thr Tyr Glu Ala Leu Thr Gln Lys Val Pro
                          485 490 495
          Gln Leu Asn Gln Met Val Arg Thr Ala Ala Glu Val Ala Gly Gln Leu
                      500 505 510
          Ile Ile Lys Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg
                  515 520 525
          Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg
              530 535 540
          Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala
          545 550 555 560
          Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly
                          565 570 575
          Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg
                      580 585 590
          Val Met Arg Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys
                  595 600 605
          Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu
              610 615 620
          Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala
          625 630 635 640
          Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr
                          645 650 655
          Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp
                      660 665 670
          Asn Glu Phe
                  675
           <![CDATA[ <210> 262]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A02]]>
           <![CDATA[ <400> 262]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
                      100 105 110
          Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro
                  115 120 125
          Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr
          145 150 155 160
          Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser
                  195 200 205
          Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Asn Met Glu Gly Ser Cys Pro Ala Arg Trp Asn Thr Asp
                      260 265 270
          Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr
                  275 280 285
          Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
                      420 425 430
          Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
                          565 570 575
          Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
              610 615 620
          Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 263]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A03]]>
           <![CDATA[ <400> 263]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
                      100 105 110
          Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro
                  115 120 125
          Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Glu Leu Tyr Thr
          145 150 155 160
          Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Glu Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser
                  195 200 205
          Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp
                      260 265 270
          Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr
                  275 280 285
          Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
                      420 425 430
          Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
                          565 570 575
          Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
              610 615 620
          Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 264]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A04]]>
           <![CDATA[ <400> 264]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
                      100 105 110
          Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro
                  115 120 125
          Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Glu Leu Tyr Thr
          145 150 155 160
          Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Glu Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser
                  195 200 205
          Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Arg Trp Lys Thr Asp
                      260 265 270
          Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr
                  275 280 285
          Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
                      420 425 430
          Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
                          565 570 575
          Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
              610 615 620
          Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 265]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A05]]>
           <![CDATA[ <400> 265]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
                      100 105 110
          Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro
                  115 120 125
          Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr
          145 150 155 160
          Ser Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Glu Ser Phe Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser
                  195 200 205
          Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp
                      260 265 270
          Ser Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr
                  275 280 285
          Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
                      420 425 430
          Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
                          565 570 575
          Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
              610 615 620
          Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 266]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A07]]>
           <![CDATA[ <400> 266]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
                      100 105 110
          Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro
                  115 120 125
          Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr
          145 150 155 160
          Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser
                  195 200 205
          Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp
                      260 265 270
          Ser Thr Cys Arg Leu Glu Thr Ser Gln Ser Lys Asn Val Lys Leu Thr
                  275 280 285
          Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
                      420 425 430
          Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
                          565 570 575
          Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
              610 615 620
          Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 267]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of A14]]>
           <![CDATA[ <400> 267]]>
          Cys Lys Gly Val Glu Pro Lys Thr Glu Cys Glu Arg Leu Ala Gly Thr
          1 5 10 15
          Glu Ser Pro Val Arg Glu Glu Pro Gly Glu Asp Phe Pro Ala Ala Arg
                      20 25 30
          Arg Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser Glu Lys Leu Asp
                  35 40 45
          Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn Glu Asn Ser Tyr
              50 55 60
          Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn Leu Ala Leu Tyr
          65 70 75 80
          Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser Lys Val Trp Arg Asp
                          85 90 95
          Gln His Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val
                      100 105 110
          Ile Ile Val Gln Ser Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro
                  115 120 125
          Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu
              130 135 140
          Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr
          145 150 155 160
          Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe
                          165 170 175
          Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu
                      180 185 190
          Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser
                  195 200 205
          Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly
              210 215 220
          Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly
          225 230 235 240
          Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys
                          245 250 255
          Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp
                      260 265 270
          Ser Thr Cys Arg Met Val Thr Ser Gln Ser Lys Asn Val Lys Leu Thr
                  275 280 285
          Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val
              290 295 300
          Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln
          305 310 315 320
          Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser Gly Val Gly Thr Ala
                          325 330 335
          Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp
                      340 345 350
          Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly
                  355 360 365
          Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser
              370 375 380
          Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val
          385 390 395 400
          Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr
                          405 410 415
          Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln
                      420 425 430
          Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu Thr
                  435 440 445
          Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala
              450 455 460
          Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro Tyr Leu Gly Thr
          465 470 475 480
          Thr Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn
                          485 490 495
          Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys
                      500 505 510
          Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn Ser
                  515 520 525
          Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile
              530 535 540
          Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp
          545 550 555 560
          Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu
                          565 570 575
          Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg
                      580 585 590
          Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro
                  595 600 605
          Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr Leu Pro Ala Leu
              610 615 620
          Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu
          625 630 635 640
          Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly
                          645 650 655
          Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe
                      660 665 670
           <![CDATA[ <210> 268]]>
           <![CDATA[ <211> 122]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_307 VH]]>
           <![CDATA[ <400> 268]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asp Lys Tyr
                      20 25 30
          Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val
              50 55 60
          Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
          65 70 75 80
          Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Thr Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Ser Gln Pro Trp Leu Tyr Arg Val Gly Val Asp Val Trp
                      100 105 110
          Gly Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 269]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_307 HCDR1]]>
           <![CDATA[ <400> 269]]>
          Lys Tyr Thr Met Asn
          1 5
           <![CDATA[ <210> 270]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_307 HCDR2]]>
           <![CDATA[ <400> 270]]>
          Val Ile Ser Asn Gly Gly Val Ser Thr Asp Tyr Ala Asp Ser Val Lys
          1 5 10 15
          Gly
           <![CDATA[ <210> 271]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of TfR1071_307 HCDR3]]>
           <![CDATA[ <400> 271]]>
          Ala Ser Gln Pro Trp Leu Tyr Arg Val Gly Val Asp Val
          1 5 10
           <![CDATA[ <210> 272]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Description of artificial sequence: amino acid sequence of CL]]>
           <![CDATA[ <400> 272]]>
          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
          
      

Claims (42)

A bispecific antibody that binds to human glypican 3 (GPC3) and human transferrin receptor (TfR). The bispecific antibody of claim 1, which binds bivalently to human GPC3 and human TfR, respectively. The bispecific antibody according to claim 1 or 2, comprising (a1) an IgG portion comprising a first antigen-binding domain and (b1) a second antigen-binding domain, the IgG portion of the first antigen-binding domain comprising the IgG portion of the heavy chain The C-terminus is linked directly or via a linker to the N-terminus of the second antigen-binding domain, either of the first antigen-binding domain and the second antigen-binding domain binds to human GPC3, and the other binds to human TfR. The bispecific antibody of claim 3, wherein the C-terminus of the heavy chain comprising the IgG portion of the first antigen-binding domain is directly linked to the N-terminus of the second antigen-binding domain. The bispecific antibody of claim 3 or 4, wherein the second antigen-binding domain is an antibody Fab. The bispecific antibody of claim 5, wherein the C-terminus of the heavy chain of the IgG portion comprising the first antigen-binding domain is linked directly or via a linker to the N-terminus of the heavy chain of the Fab of the antibody. The bispecific antibody of claim 1 or 2, comprising (a2) an IgG portion comprising a second antigen-binding domain and (b2) a first antigen-binding domain, the C-terminus of the first antigen-binding domain directly or via a linker Linked to the N-terminus of the heavy chain comprising the IgG portion of the second antigen-binding domain, either the first antigen-binding domain or the second antigen-binding domain binds to human GPC3, and the other binds to human TfR. The bispecific antibody of claim 7, wherein the C-terminus of the first antigen-binding domain is directly linked to the N-terminus of the heavy chain of the IgG portion of the second antigen-binding domain. The bispecific antibody of claim 7 or 8, wherein the first antigen-binding domain is an antibody Fab. The bispecific antibody of claim 9, wherein the C-terminus of the heavy chain of the Fab of the antibody is linked directly or via a linker to the N-terminus of the heavy chain of the IgG portion comprising the second antigen-binding domain. The bispecific antibody according to any one of claims 3 to 10, wherein any one of the first antigen-binding domain and the second antigen-binding domain is an anti-human GPC3 IgG antibody Fab (anti-GPC3 Fab). The bispecific antibody according to any one of claims 3 to 11, wherein any one of the first antigen-binding domain and the second antigen-binding domain is an anti-human TfR IgG antibody Fab (anti-TfR Fab). The bispecific antibody according to any one of claims 3 to 12, wherein the first antigen-binding domain is an anti-GPC3 Fab, and the second antigen-binding domain is an anti-TfR Fab. The bispecific antibody according to any one of claims 3 to 12, wherein the first antigen-binding domain is an anti-TfR Fab, and the second antigen-binding domain is an anti-GPC3 Fab. The bispecific antibody according to any one of claims 11 to 14, wherein the above-mentioned anti-GPC3 Fab is a Fab comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region ( VH) contains any one of complementarity determining regions (CDRs) 1 to 3 selected from the following (g1) to (g20), (g23) and (g25) to (g40), and the light chain variable region (VL) contains CDR1-3 respectively comprising the amino acid sequences represented by SEQ ID NOs: 24-26, (g1) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 91 to 93, (g2) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 95 to 97, (g3) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 99 to 101, (g4) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 103 to 105, (g5) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 107 to 109, (g6) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 111 to 113, (g7) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 115 to 117, (g8) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 119 to 121, respectively, (g9) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 123 to 125, (g10) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 127 to 129, (g11) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 131 to 133, (g12) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 135 to 137, (g13) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 139 to 141, (g14) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 143 to 145, (g15) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 147 to 149, (g16) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 151 to 153, (g17) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 155 to 157, (g18) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 159 to 161, (g19) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 163 to 165, (g20) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 167 to 169, (g23) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 179 to 181, (g25) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 187 to 189, (g26) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 191 to 193, (g27) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 195 to 197, (g28) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 199 to 201, (g29) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 203 to 205, (g30) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 207 to 209, (g31) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 211 to 213, (g32) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 215 to 217, (g33) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 219 to 221, (g34) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 223 to 225, (g35) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 227 to 229, (g36) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 231 to 233, (g37) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 235 to 237, respectively, (g38) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 239 to 241, respectively, (g39) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 243 to 245, respectively, (g40) CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 247 to 249, respectively. The bispecific antibody according to any one of claims 11 to 15, wherein the above-mentioned anti-GPC3 Fab comprises a compound selected from the group consisting of SEQ ID NOs: 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134,138,142,146,150,154,158,162,166,178,186,190,194,198,202,206,210,214,218,222,226,230,234,238,242 and The VH of the amino acid sequence represented by any one of 246, and the Fab comprising the VL of the amino acid sequence represented by SEQ ID NO: 23. The bispecific antibody according to any one of claims 12 to 16, wherein the anti-TfR Fab is a Fab comprising VH and VL, and the VH comprises CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 28 to 30, respectively, The VL contains CDRs 1 to 3 containing the amino acid sequences represented by SEQ ID NOs: 24 to 26, respectively. The bispecific antibody according to any one of claims 12 to 16, wherein the anti-TfR Fab is a Fab comprising VH and VL, and the VH comprises CDRs 1 to 3 comprising the amino acid sequences represented by SEQ ID NOs: 41 to 43, respectively, The VL contains CDRs 1 to 3 containing the amino acid sequences represented by SEQ ID NOs: 24 to 26, respectively. The bispecific antibody according to any one of claims 12 to 17, wherein the anti-TfR Fab is a VH comprising the amino acid sequence represented by SEQ ID NO: 35 and VL comprising the amino acid sequence represented by SEQ ID NO: 23 Fab. The bispecific antibody according to any one of claims 12 to 16 and 18, wherein the anti-TfR Fab is a VH comprising the amino acid sequence represented by SEQ ID NO: 40 and a VH comprising the amino acid sequence represented by SEQ ID NO: 23 Fab of VL. The bispecific antibody according to any one of claims 3 to 20, wherein the IgG portion comprising the first antigen-binding domain or the IgG portion comprising the second antigen-binding domain comprises an amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 255 Heavy chain constant region. The bispecific antibody according to any one of claims 1 to 6, 11 to 13, 15 to 17, 19 and 21, comprising 2 heavy chains and 4 light chains, the 2 heavy chains respectively starting from the N-terminus The VH comprising the amino acid sequence represented by any one of SEQ ID NOs: 90, 142, 126, 178, 198, 146, 218, 150, 202, and 226, and the amino acid represented by SEQ ID NO: 255 are contained in this order CH of the sequence, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, the four light chains respectively contain the amine represented by SEQ ID NO: 23 in sequence from the N-terminus VL of the amino acid sequence and CL comprising the amino acid sequence represented by SEQ ID NO: 272. The bispecific antibody of any one of claims 1, 2, 7 to 13, 15 to 17, 19 and 21, comprising 2 heavy chains and 4 light chains, the 2 heavy chains respectively starting from the N-terminus Sequentially contains a sequence number 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 178, VH of the amino acid sequence represented by any one of 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246, including SEQ ID NO: 253 The CH1 of the amino acid sequence, the VH comprising the amino acid sequence represented by SEQ ID NO: 35, and the CH comprising the amino acid sequence represented by SEQ ID NO: 255, the four light chains from the N-terminus respectively contain the sequence number comprising SEQ ID NO: 255. VL of the amino acid sequence represented by 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272. The bispecific antibody of any one of claims 1, 2, 7 to 13, 15, 16, 18, 20, and 21, comprising 2 heavy chains and 4 light chains, the 2 heavy chains being respectively N The terminus sequentially contains a sequence number selected from the group consisting of: VH of the amino acid sequence represented by any one of 178, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242 and 246, including SEQ ID NO: CH1 of the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 40, and CH comprising the amino acid sequence represented by SEQ ID NO: 255; VL of the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272. The bispecific antibody of claim 24, comprising 2 heavy chains and 4 light chains, the two heavy chains from the N-terminus respectively contain a sequence selected from the group consisting of SEQ ID NOs: 94, 98, 102, 106, 114, VH of the amino acid sequence represented by any one of 130, 178 and 190, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 40, and represented by SEQ ID NO: 255 The CH of the amino acid sequence, the four light chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 from the N-terminus, respectively. The bispecific antibody according to claim 24, comprising 2 heavy chains and 4 light chains, the 2 heavy chains from the N-terminus respectively contain a sequence selected from the group consisting of SEQ ID NOs: 94, 98, 102, 114, 130, VH of the amino acid sequence represented by any one of 166, 178, 186 and 190, CH1 comprising the amino acid sequence represented by SEQ ID NO: 253, VH comprising the amino acid sequence represented by SEQ ID NO: 35, and SEQ ID NO: 35 CH of the amino acid sequence represented by 255, the four light chains respectively contain VL comprising the amino acid sequence represented by SEQ ID NO: 23 and CL comprising the amino acid sequence represented by SEQ ID NO: 272 from the N-terminus, respectively. The bispecific antibody of any one of claims 1 to 26, which has cell proliferation inhibitory activity. The bispecific antibody of any one of claims 1 to 27, which does not inhibit the proliferation of cells that do not express GPC3 and express TfR, but inhibits the proliferation of cells that co-express GPC3 and TfR. A bispecific antibody fragment, which is a bispecific antibody fragment of the bispecific antibody of any one of claims 1 to 28. A nucleic acid comprising a base sequence encoding the bispecific antibody according to any one of claims 1 to 28 or the bispecific antibody fragment according to claim 29. A recombinant vector containing the nucleic acid of claim 30. A transformant is obtained by introducing the recombinant vector of claim 31 into a host cell. A method for producing the bispecific antibody as claimed in any one of claims 1 to 28 or the bispecific antibody fragment as claimed in claim 29, characterized in that the transformed strain as claimed in claim 32 is cultured in a culture medium, and the cultured The bispecific antibody as claimed in any one of claims 1 to 28 or the bispecific antibody fragment as claimed in claim 29 is produced and accumulated in the culture, and the bispecific antibody or the bispecific antibody fragment is collected from the culture. A therapeutic drug and/or diagnostic drug for a disease related to at least one of GPC3 and TfR, which contains the bispecific antibody according to any one of claims 1 to 28 or the bispecific antibody fragment according to claim 29 as effective Element. The therapeutic drug and/or diagnostic drug of claim 34, wherein the disease associated with at least one of GPC3 and TfR is cancer. A method for treating and/or diagnosing a disease associated with at least one of GPC3 and TfR, using the bispecific antibody according to any one of claims 1 to 28 or the bispecific antibody fragment according to claim 29. The method of treatment and/or diagnosis of claim 36, wherein the disease associated with at least one of GPC3 and TfR is cancer. The bispecific antibody according to any one of claims 1 to 28 or the bispecific antibody fragment according to claim 29 for use in the treatment and/or diagnosis of a disease associated with at least one of GPC3 and TfR. The bispecific antibody or bispecific antibody fragment of claim 38, wherein the disease associated with at least one of GPC3 and TfR is cancer. Use of the bispecific antibody according to any one of claims 1 to 28 or the bispecific antibody fragment according to claim 29 for the manufacture of a therapeutic drug and/or a disease associated with at least one of GPC3 and TfR Diagnostic medicine. The use of claim 40, wherein the disease associated with at least one of GPC3 and TfR is cancer. A reagent for detecting or measuring at least one of GPC3 and TfR, comprising a bispecific antibody as claimed in any one of claims 1 to 28 or a bispecific antibody fragment as claimed in claim 29.

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