RU2810756C2 - Bispecific antibodies binding to tfr - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a bispecific antibody or a fragment of this bispecific antibody that binds to the transferrin receptor (TfR) and to a cell surface antigen, as well as to a method of its preparation. Also the following is disclosed: the DNA encoding the above antibody or a fragment thereof, as well as the vector and transformant containing it.

EFFECT: invention is effective for treating a disease associated with at least one of human TfR and EGFR, which is a cancer.

18 cl, 21 dwg, 7 tbl, 20 ex

Description

Field of technology to which the invention relates

This invention relates to bispecific antibodies containing an antigen-binding domain that binds to the transferrin receptor (TfR), and an antigen-binding domain that binds to a cell surface antigen; to bispecific antibody fragments; to DNA encoding said bispecific antibodies or bispecific antibody fragments; to vectors comprising said DNA; to hybridomas or transformed cells producing said bispecific antibodies or bispecific antibody fragments; to a method for producing said bispecific antibodies or bispecific antibody fragments; to therapeutic or diagnostic agents including these bispecific antibodies or bispecific antibody fragments; to methods of treatment and diagnosis that use said bispecific antibodies or bispecific antibody fragments, and to detection and quantitation reagents that include said bispecific antibodies or bispecific antibody fragments.

Prior Art

Antibodies are glycoproteins present in the serum or tissue fluid of all mammals that recognize foreign antigens in the body. Antibodies participate in host defense by activating the complement system or effector functions of FcR-expressing cells, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), mediator release, and antigen presentation through binding to a cell surface receptor (FcR).

The antibody molecule consists of four polypeptide chains: two homologous light (L) and two homologous heavy (H) and contains two antigen-binding domains. Antibodies are divided into classes and subclasses based on their heavy chains; Each class and subclass has specific functions. There are five different classes of human antibodies: IgG, IgA, IgM, IgD and IgE. IgG is further divided into the subclasses IgG1, IgG2, IgG3, and IgG4; and IgA is further divided into the subclasses IgA1 and IgA2 (Charles A. J. et. al., Immunobiology, 1997, Current Biology Ltd/Garland Publishing, Inc.).

A polyvalent antibody is an antibody whose molecule contains several antigen-binding domains. An example of a multivalent antibody is the first described bivalent antibody that binds monovalently to each of two different antigens, which was produced by expressing in one cell H and L chains derived from two different types of antibodies using hybridoma technology (NPL 1). However, this method produces about ten combinations of the H and L chains of an antibody. Therefore, the amount of multivalent antibody with the desired combination of L and H chains is small, and furthermore, it is difficult to selectively isolate and purify the desired multivalent antibody, so that the yield of the desired antibody is low.

To overcome these difficulties, attempts have been made to produce an antibody with the desired combination by combining several antigen-binding domains and expressing them as a single polypeptide chain to reduce the number of subunit combination options.

For example, scFv antibodies are known that comprise a single Fv polypeptide chain in which the antigen-binding domains of the L and H chains are combined into a single polypeptide (NPL 2). Antibodies have also been reported that combine two antigen-binding domains using the CH1 domain of the IgG1 heavy chain constant region or a partial fragment of this domain, the light chain constant region or a flexible linker (Gly-Gly-Gly-Gly-Ser), and others similar antibodies (PTL1, PTL2).

These conventional multivalent antibodies have the disadvantage that they are prone to aggregation, and the stability and productivity of their production are low. However, it has been found that those multivalent antibodies which include multiple antigen-binding domains within a single heavy chain polypeptide and in which the heavy chain variable regions are connected to each other by a linker with the amino acid sequence of an immunoglobulin domain or fragment thereof are highly stable and can be produced with high productivity (PTL 3).

The transferrin receptor (also referred to herein as TfR) has been identified as a cell membrane component expressed on the surface of reticulocytes. The functions of TfR include the transfer of iron atoms bound to transferrin (herein referred to as Tf) into the cell. Iron is critical for maintaining cellular homeostasis and for cell proliferation, so for iron uptake, the transferrin receptor is expressed at high levels in the trophoblast cells of the placenta, in reticulocytes, in activated lymphocytes and in those tissues whose cells normally absorb a lot of iron. It is also known that the expression level of TfR is high in various actively proliferating tumor cells (NPL 3 and 4). It is also known that when TfR is cross-linked to the cell membrane, the resulting complex is internalized by clathrin-dependent endocytosis and subsequent breakdown of TfR (NPL 5 and 6).

Since the expression level of TfR is low in normal cells, with the exception of bone marrow cells, and high in actively proliferating cancer cells, the transferrin receptor has long been considered as a molecular target for the treatment of cancer. A TfR-targeting molecule, such as a transferrin receptor neutralizing antibody, has been shown to be highly effective as a drug in mouse models of cancer (PTL 4).

It is known that cancer cells have increased expression of certain membrane proteins compared to normal cells. Drugs have been developed that kill only cancer cells using antibodies that selectively recognize such membrane proteins. For example, antibody drugs targeting the epidermal growth factor receptor (hereinafter referred to as EGFR) have been shown to be effective in colon cancer or head and neck cancer (NPL 7).

EGFR is a tyrosine kinase-type receptor found as a receptor for epidermal growth factor (hereinafter referred to as EGF). EGFR is a transmembrane glycoprotein with a molecular weight of 170 kDa; when it binds its ligand, it forms a dimer and activates the subsequent cascade of intracellular signaling pathways, thereby promoting cell proliferation, differentiation, tissue invasion, metastasis, etc.

EGFR is expressed in a variety of epithelial, mesenchymal and neural cells, but is expressed at high levels in many cancer cells, such as brain tumors and squamous cell carcinomas (NPL 8 and 9); thus, EGFR is considered a tumor antigen.

It has been established that if a molecule is used that binds to two different membrane proteins, then one antigen molecule can serve as a core structure and the second antigen can be internalized and subject to degradation (PTL 5 and 6). It has also been reported that if a peptide that recognizes a cancer cell-specific membrane protein and a peptide that recognizes TfR are chemically linked or fused using a fusion protein technique, then in the presence of an iron chelating agent, there is a growth inhibition of cells in which they are expressed. both of these antigens (PTL 7).

List of bibliographic references

Patent publications

PTL 1 - US Patent Application No. 2007/0071675

PTL 2 - WO 2001/077342

PTL 3 - WO 2009/131239

PTL 4 - WO 2012/153707

PTL 5 - WO 2013/138400

PTL 6 - European Patent Application No. 2808035

PTL 7 - US Patent No. 57025614

Non-patent publications

NPL 1 - Suresh M.R. et. al., Methods Enzymol. 121, 210-228, 1986

NPL 2 – Kranz D.M. et. al., J. Hematother. 5, 403-408, 1995

NPL 3 - Hamilton T.A. et al., PNAS USA 76 6406-10, 1979

NPL 4 - Larson S.M. et al., J. Natl. Cancer Inst. 64 41-53, 1980

NPL 5 - Liu A.P. et al., J. Cell Biol. 191 1381-93, 2010

NPL 6 - Weissman A.M. et al., J. Cell Biol. 102 951-8, 1986

NPL 7 – Instructions for use of USA-made Erbitux (cetuximab) (as amended June 2018)

NPL 8 - Libermann T.A. et al., Cancer Res. 44 753-60, 1984

NPL 9 - Hendler F.J. et al., J. Clin. Invest. 74 647-51, 1984

Disclosure of the Invention

Technical problem

The objective of this invention is to provide new bispecific antibodies that bind to TfR and cell surface antigen; bispecific antibody fragments; DNA encoding the specified bispecific antibodies or bispecific antibody fragments; vectors comprising said DNA; hybridomas or transformed cells producing said bispecific antibodies or bispecific antibody fragments; a method for producing said bispecific antibodies or bispecific antibody fragments; therapeutic or diagnostic agents including these bispecific antibodies or bispecific antibody fragments; methods of treatment and diagnostics that use said bispecific antibodies or bispecific antibody fragments, and a detection or quantitation reagent comprising said bispecific antibodies or bispecific antibody fragments.

Solving a technical problem

To solve the above object, the present invention provides a bispecific antibody or bispecific antibody fragment that binds to TfR and to a cell surface antigen and in which a polypeptide containing a domain that binds a cell surface antigen directly (also called an N-terminal side polypeptide) is directly linked or through a linker to the N-terminal side of the portion of the IgG molecule that binds to TfR.

So, this invention relates to the following:

1. A bispecific antibody or bispecific antibody fragment that binds to TfR (TfR) and to a cell surface antigen and includes a portion of the IgG molecule that binds to TfR and an N-terminal polypeptide that binds to a cell surface antigen, wherein said N polypeptide -terminal side directly or through a linker structure is linked to the N-terminus of the heavy chain of the specified IgG part.

2. A bispecific antibody or bispecific antibody fragment according to paragraph 1 above, wherein the IgG portion includes a heavy chain variable region (VH) containing

- a complementarity determining region (CDR) 1, which includes the amino acid sequence represented by SEQ ID NO: 32, or an amino acid sequence that is the product of modification of the sequence SEQ ID NO: 32 by at least one amino acid substitution selected from tyrosine substitutions at position 2 to alanine or phenylalanine and threonine at position 3 to alanine or glycine;

- a CDR2 region that includes the amino acid sequence represented by SEQ ID NO: 33, or an amino acid sequence that is the product of modification of the sequence SEQ ID NO: 33 by at least one amino acid substitution selected from substitutions of valine at position 1 with alanine, isoleucine at position 2 for alanine or leucine, valine at position 7 for glutamic acid, aspartic acid at position 10 for alanine, and aspartic acid at position 13 for proline; And

a CDR3 region that includes the amino acid sequence represented by SEQ ID NO: 34, or an amino acid sequence that is the product of modification of the sequence SEQ ID NO: 34 by at least one amino acid substitution selected from substitutions of glutamine at position 3 with alanine or aspartic acid, proline at position 4 to a randomly selected natural amino acid, tryptophan at position 5 to alanine, phenylalanine, histidine or tyrosine, tyrosine at position 7 to alanine or phenylalanine, and valine at position 13 to leucine; And

a light chain variable region (VL) having CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 17 - 19, respectively.

3. A bispecific antibody or a bispecific antibody fragment according to item 1 above, wherein the IgG portion includes one heavy chain variable region (VH) selected from items (ai) to (ci) below and a light chain variable region (VL) having . CDRs 1–3, containing the amino acid sequences represented by SEQ ID NO: 17–19, respectively;

(ai) VH containing CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 32-34, respectively;

(bi) VH containing CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 42-44, respectively;

(ci) VH containing CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 47 - 49, respectively.

4. A bispecific antibody or bispecific antibody fragment according to paragraph 1 or 2 above, wherein the IgG portion includes a VH containing an amino acid sequence represented by any of the sequences of SEQ ID NOs: 26, 31, 36, 41 and 46, and a VL containing an amino acid sequence represented by SEQ ID NO: 16.

5. A bispecific antibody or a bispecific antibody fragment according to paragraph 1 above, wherein the IgG portion includes a VL containing CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 17-19, respectively, and one heavy chain variable region (VH), selected from (a) - (m) below:

(a) VH containing CDRs 2 and 3, containing the amino acid sequences represented by SEQ ID NO: 33 and 34, respectively, and CDR1, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 32 by replacing the tyrosine at position 2 with alanine or phenylalanine;

(b) VH containing CDRs 2 and 3, containing the amino acid sequences represented by SEQ ID NO: 33 and 34, respectively, and CDR1, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 32 by replacing threonine at position 3 with alanine or glycine;

(c) VH containing CDRs 1 and 3, containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 33 by replacing valine at position 1 with alanine;

(d) VH containing CDRs 1 and 3, containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 33 by replacing isoleucine at position 2 with alanine or leucine;

(e) VH containing CDRs 1 and 3 containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2 which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 33 by replacing valine at position 7 with glutamic acid;

(f) VH containing CDRs 1 and 3, containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 33 by replacing aspartic acid at position 10 with alanine ;

(g) VH containing CDRs 1 and 3, containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 33 by replacing aspartic acid at position 13 with proline ;

(h) a heavy chain variable region with CDRs 1 and 2 comprising the amino acid sequences represented by SEQ ID NOs: 32 and 33, respectively, and CDR3 which includes the amino acid sequence resulting from modification of the sequence SEQ ID NO: 34 by substitution of glutamine at position 3 for alanine or aspartic acid;

(i) VH containing CDRs 1 and 2, containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3, which includes an amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 34 by replacing the proline at position 4 with an arbitrary one natural amino acid;

(j) VH containing CDRs 1 and 2, containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 34 by replacing the proline at position 4 with alanine, tyrosine, serine, aspartic acid, glutamine, glutamic acid, threonine, arginine, glycine, lysine, methionine, valine, leucine, isoleucine, tryptophan, phenylalanine or histidine;

(k) VH containing CDRs 1 and 2, containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 34 by replacing tryptophan at position 5 with alanine, phenylalanine, histidine or tyrosine;

(l) VH containing CDRs 1 and 2, containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 34 by replacing the tyrosine at position 7 with alanine or phenylalanine; And

(m) VH containing CDRs 1 and 2, containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3, which includes the amino acid sequence that is the product of modification of the sequence of SEQ ID NO: 34 by replacing valine with leucine.

6 The bispecific antibody or bispecific antibody fragment according to the above paragraph 1, wherein the IgG part includes a VL containing the amino acid sequence represented by SEQ ID NO: 16, and a VH containing an amino acid sequence being the product of a modification of the amino acid sequence. represented by SEQ ID NO: 31 by at least one amino acid residue substitution selected from substitutions Y32A, Y32F, T33A, T33G, L45A, V48A, V50A, I51A, I51L, V55E, D58A, D61P, Q97A, Q97D, W99A, W99F, W99H, W99Y, Y100AA, Y100AF, V102L and replacement of P98 with a randomly selected amino acid (amino acid residue numbering is indicated in the EU numbering system consistent with the Cabot system (Kabat E.A. et al. 1991) hereinafter referred to as the “EU index”).

7. The bispecific antibody or bispecific antibody fragment according to paragraph 1 above, wherein the IgG portion includes a VL containing the amino acid sequence represented by SEQ ID NO: 16, and a VH containing an amino acid sequence being the product of a modification of the amino acid sequence. represented by SEQ ID NO: 31 by at least one amino acid residue substitution selected from substitutions Y32A, Y32F, T33A, T33G, L45A, V48A, V50A, I51A, I51L, V55E, D58A, D61P, Q97A, Q97D, W99A, W99F, W99H, W99Y, Y100AA, Y100AF, V102L, P98A, P98Y, P98S, P98D, P98Q, P98E, P98T, P98R, P98G, P98K, P98M, P98V, P98L, P98I, P98W, P98F and P98H (EU numbering).

8. The bispecific antibody or bispecific antibody fragment as set forth in 1 above, wherein the IgG portion recognizes at least one amino acid residue selected from the residues Asp at position 352, Ser at position 355, Asp at position 356, and Lys at position 358, and at least one amino acid residue selected from Met at position 365, Val at position 366, and Glu at position 369 of the TfR amino acid sequence represented by SEQ ID NO: 6.

9. The bispecific antibody or bispecific antibody fragment according to any one of paragraphs 1 to 8 above, wherein the heavy chain constant region of the IgG portion contains the amino acid sequence represented by SEQ ID NO: 84 or SEQ ID NO: 86.

10. A bispecific antibody or bispecific antibody fragment according to any one of paragraphs 1 to 9 above, wherein the cell surface antigen is EGFR or GPC.

11. A bispecific antibody or bispecific antibody fragment according to any one of paragraphs 1 to 10 above, wherein the cell surface antigen is EGFR.

12. The bispecific antibody or bispecific-specific antibody fragment of any one of paragraphs 1-11 above, wherein the N-terminal side polypeptide is any of the Fab, Fab', scFv, dsFv and VHH fragments of an antibody directed against the cell surface antigen.

13. A bispecific antibody or bispecific antibody fragment according to any one of paragraphs 1-12 above, wherein the N-terminal side polypeptide is a Fab fragment of an antibody directed against a given cell surface antigen.

14. A bispecific antibody or bispecific antibody fragment according to any one of paragraphs 1-9 above, wherein the N-terminal side polypeptide is a Fab fragment of an anti-EGFR antibody, and the antibody is an antibody comprising a VH containing CDRs 1-3 containing amino acid sequences, represented by SEQ ID NOs: 60-62, respectively, or SEQ ID NOs: 65-67, respectively, and VL containing CDRs 1-3 containing the amino acid sequences represented by SEQ ID NOs: 17-19, respectively.

15. The bispecific antibody or bispecific antibody fragment of paragraph 14 above, wherein the anti-EGFR antibody is an antibody selected from those described in paragraphs (a) to (e):

(a) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 59 or SEQ ID NO: 64, and a VL containing the amino acid sequence represented by SEQ ID NO: 16;

(b) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 110, and a VL containing the amino acid sequence represented by SEQ ID NO: 111;

(c) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 112, and a VL containing the amino acid sequence represented by SEQ ID NO: 113;

(d) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 114, and a VL containing the amino acid sequence represented by SEQ ID NO: 115; And

(e) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 116 and a VL containing the amino acid sequence represented by SEQ ID NO: 117.

16. The bispecific antibody or bispecific antibody fragment according to any one of paragraphs 13-15 above, wherein the C-terminus of the heavy chain of the Fab fragment is directly connected to the N-terminus of the heavy chain of the IgG portion.

17. The bispecific antibody or bispecific antibody fragment according to any one of paragraphs 13 to 15 above, wherein the C-terminus of the heavy chain of the Fab fragment is linked to the N-terminus of the heavy chain of the IgG portion via a linker.

18. The bispecific antibody or bispecific antibody fragment according to paragraph 17 above, wherein the amino acid sequence of the linker consists of all or part of the amino acid sequence of the hinge region of the IgG molecule.

19. The bispecific antibody or bispecific antibody fragment according to any one of paragraphs 13 to 18 above, wherein the amino acid sequence constituting the light chain in the Fab fragment and the amino acid sequence constituting the light chain in the IgG portion are the same.

20. DNA encoding a bispecific antibody or a bispecific antibody fragment according to any one of paragraphs 1-19.

21. Recombinant vector containing DNA according to paragraph 20 above.

22. A transformed cell obtained by introducing the recombinant vector according to paragraph 21 above into the host cell.

23. A method for producing a bispecific antibody or a bispecific antibody fragment according to any of the above paragraphs 1-19, including cultivating the transformed cells according to paragraph 22 in a culture medium that ensures that these cells produce and accumulate in their culture a bispecific antibody or a bispecific antibody fragment according to any of paragraphs 1-19, as well as the isolation of the specified bispecific antibody or bispecific antibody fragment from this culture.

24. A therapeutic and/or diagnostic agent used for diseases associated with at least one of a human TfR or a cell surface antigen, wherein said agent comprises as an active ingredient a bispecific antibody or a bispecific antibody fragment according to any one of claims 1-19.

25. The therapeutic and/or diagnostic agent as set forth in claim 24 above, wherein the disease associated with at least one of the human TfR or cell surface antigen is a cancer.

26. A method of treating and/or diagnosing a disease associated with at least one of a human TfR or a cell surface antigen, which uses a bispecific antibody or bispecific antibody fragment according to any of paragraphs 1-19 above.

27. The method of claim 26 above, wherein the disease with at least one of a human TfR or a cell surface antigen is a cancer.

28. A bispecific antibody or bispecific antibody fragment according to any one of paragraphs 1-19 above for use in the treatment or diagnosis of a disease associated with at least one of a human TfR or a cell surface antigen.

29. The bispecific antibody or bispecific antibody fragment of claim 28 above, wherein the disease associated with at least one of the human TfR or cell surface antigen is a cancer.

30. Use of a bispecific antibody or bispecific antibody fragment according to any of paragraphs 1-19 above to produce a therapeutic or diagnostic agent associated with at least one of a human TfR or a cell surface antigen.

31. The use of claim 30 above, wherein the disease associated with at least one of the human TfR or cell surface antigen is a cancer.

32. A reagent for detecting or quantifying at least one of a human TfR or a cell surface antigen, containing a bispecific antibody or bispecific antibody fragment according to any of paragraphs 1-19 above

Beneficial effects of the invention

This invention provides new bispecific antibodies and bispecific antibody fragments that bind to TfR and cell surface antigen; DNA encoding said bispecific antibodies and bispecific antibody fragment; vectors containing said DNA; hybridomas and transformed cells producing these bispecific antibodies and bispecific antibody fragments; a method for producing said bispecific antibodies and bispecific antibody fragments; therapeutic and diagnostic agents including these bispecific antibodies and bispecific antibody fragments; methods of treatment and diagnosis using said bispecific antibodies and bispecific antibody fragments, and detection and quantitation reagents containing said bispecific antibodies and bispecific antibody fragments.

Brief description of drawings

Figure 1 depicts an example of the structure of a bispecific antibody according to this invention. In fig. 1(A) shows a bispecific antibody in which the N-terminal side polypeptide is Fab and the polypeptide comprising the Fab heavy chain variable region is linked to the N-terminal side of the IgG portion. In fig. 1(B) shows a bispecific antibody in which the N-terminal side polypeptide is VHH. In fig. 1(C) shows a bispecific antibody in which the N-terminal side polypeptide is a Fab and a polypeptide comprising a Fab light chain variable region is linked to the N-terminal side of an IgG portion.

Figure 2 presents the results of determining the level of expression of a specific antigen in cancer cells of various lines by flow cytometry. In fig. 2(A)-2(C) show the results of determining the level of EGFR expression in OE21, T.Tn and U-937 cells, respectively, using a flow cytometer. In fig. 2(D)-2(F) show the results of determining the expression level of TfR (TfR) in OE21, T.Tn and U-937 cells, respectively, by flow cytometer. The ordinate axis is the number of cells, the abscissa axis is the fluorescence intensity. The solid line represents the binding affinity of anti-TfR antibody or anti-EGFR antibody; The gray histogram represents the binding affinity of the isotype antibody that served as a negative control.

Figure 3 presents the results of determining the expression level of various antigens in liver cancer cells by flow cytometry. In fig. 3(A)-3(C) show the results of determining the expression level of GPC3 in HepG2, HuH-7 and HLE cells, respectively, by flow cytometer. In fig. 3(D)-3(F) - results of determining the level of TfR expression in HepG2, HuH-7 and HLE cells, respectively, using a flow cytometer. In fig. 3(G)-3(I) - results of determining the level of EGFR expression in HepG2, HuH-7 and HLE cells, respectively, using a flow cytometer. The ordinate axis is the number of cells, the abscissa axis is the fluorescence intensity. The solid line represents the binding affinity of antibodies to GPC3 or TfR or EGFR; The gray histogram represents the binding affinity of the isotype antibody (or anti-DNP antibody serving as a negative control.

Figure 4 presents the results of determining the binding affinity of various antibodies (A)-(I) to OE21 cells by flow cytometry. The ordinate axis is the number of cells, the abscissa axis is the fluorescence intensity. The solid line represents the binding affinity of anti-TfR or anti-EGFR antibodies; The gray histogram represents the binding affinity of the secondary antibodies that served as negative controls.

Figure 5 presents the results of determining the growth inhibition of OE21 cells by various anti-TfR antibodies. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control. Black bars - only monoclonal antibodies were added, white bars - cross-linking antibodies were added, connecting monoclonal antibody molecules to each other.

Figure 6 presents the results of determining the inhibition of OE21 cell growth by each of the anti-EGFR antibodies. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 7A presents the results of determining the growth inhibition of OE21 cells by various bispecific antibodies. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 7B presents the results of determining the growth inhibition of OE21 cells by various bispecific antibodies. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 7C presents the results of determining the inhibition of OE21 cells by various bispecific antibodies. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 8A presents the results of determining the growth inhibition of the EGFR-expressing cancer cell line OE21 by an EGFR-TfR bispecific antibody. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 8B shows the results of determining the growth inhibition of the T.Tn cancer cell line that expresses EGFR by an EGFR-TfR bispecific antibody. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 8C shows the results of growth inhibition of the EGFR-expressing cancer cell line U-937 by an EGFR-TfR bispecific antibody. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 9 presents the results of determining the growth inhibition of HepG2 cells by GPC3-TfR bispecific antibodies. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 10A presents the results of growth inhibition of HepG2 cells, which is a liver cancer cell line expressing GPC3, by a GPC3-TfR bispecific antibody. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 10B presents the results of determining the growth inhibition of HuH-7 cells, which is a liver cancer cell line that expresses GPC3, by a GPC3-TfR bispecific antibody. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 10C presents the results of HLE cell growth inhibition determination. which is a liver cancer cell line that expresses GPC3, a GPC3-TfR bispecific antibody. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control.

Figure 11 (A-E) presents the results of determining the inhibition of TfR binding to transferrin by various antibodies or bispecific antibodies on OE21 cells. The ordinate axis is the number of cells, the abscissa axis is the fluorescence intensity. The thin solid line is the binding activity under conditions where fluorescently labeled transferrin was added after the addition of the negative control. The thick solid line is the binding activity under conditions where fluorescently labeled transferrin was added after the addition of antibodies or bispecific antibodies. The histogram shaded in gray is the control (without the addition of fluorescently labeled transferrin).

Figure 12 presents the results of determining the effect on EGFR downstream signaling and on the level of TfR expression when adding EGFR-TfR bispecific antibodies to OE21 cells by Western blotting. Each bar represents the expression level of each protein listed in the vertical line on the right when antibodies or bispecific antibodies were added at the concentrations indicated at the top. pEGFR – phosphorylated EGFR; AKT – EGFR signaling pathway protein; pAKT - phosphorylated AKT; TFRC – transferrin receptor; ACTB - β-actin.

Figure 13. (A-D) presents the results of flow cytometry determination of the effect of adding EGFR-TfR bispecific antibodies to OE21 cells on the level of TfR expression on the cell surface. The ordinate axis is the number of cells, the abscissa axis is fluorescence intensity. Thin solid line is activity binding under conditions where fluorescently labeled transferrin was added after the addition of a negative control and culture for 24 hours. The thick solid line is the binding activity under conditions where fluorescently labeled transferrin was added after the addition of antibodies or bispecific antibodies and culture for 24 hours. The histogram shaded in gray is the control (without the addition of fluorescently labeled transferrin).

Figure 14 presents the results of determining whether intracellular iron depletion affects the inhibition of OE21 cell growth by EGFR-TfR bispecific antibodies. The y-axis is cell viability, expressed as the ratio of the ATP level in living cells to its level in the negative control under conditions where PBS was added and no FAS was added. Black bars - only antibody or bispecific antibody was added; Filled bars—FAS was added along with the antibody or bispecific antibody.

Figure 15 presents the results of determining the inhibition of OE21 cell growth upon addition of EGFR-TfR bispecific antibodies. The ordinate is the relative content (%) of cells in the well. Black bars – the relative content (%) of cells in a well during cultivation for 7 days in the presence of an antibody or bispecific antibody. The shaded bars are the relative content (%) of cells in the well during cultivation for 7 days in the presence of an antibody or bispecific antibody, and subsequent removal of the antibody by changing the medium and culturing for another 7 days.

Figure 16 presents the results of the GEMM growth inhibition determination. with the addition of EGFR-TfR bispecific antibodies. The ordinate is cell viability, where the level of ATP in viable cells upon addition of PBS was taken as 100%.

Figure 17A presents the results of determining cell growth inhibition by various EGFR-TfR bispecific antibodies.

Figure 17B presents the results of determining the inhibition of cell growth by various EGFR-TfR bispecific antibodies.

Figure 18 (A) schematically depicts the structure of EGFR-TfR bispecific antibodies prepared in Example 6, and (B) schematically depicts the structure of heterodimeric antibodies prepared in Example 23.

Figure 19A presents the results of cell growth inhibition assays with various EGFR-TfR bispecific antibodies.

Figure 19B presents the results of cell growth inhibition assays with various EGFR-TfR bispecific antibodies.

Figure 20 presents the results of determining the inhibition of cell growth by various EGFR-TfR bispecific antibodies, as indicated by the inhibition of the growth of a cancer cell line

Figure 21 presents the results of determining the inhibition of cell growth by various EGFR-TfR bispecific antibodies.

Description of embodiments of the invention

This invention relates to a bispecific antibody or bispecific antibody fragment that binds to TfR and to a cell surface antigen, and in which a polypeptide (also called an N-terminal side polypeptide) containing an antigen-binding domain specific for a cell surface antigen is linked directly or via a linker to the N-terminal side of the portion of the IgG that binds to the TfR (hereinafter these antibodies and antibody fragments are referred to as bispecific antibodies and bispecific antibody fragments of this invention).

In this document, the designation TfR is used as a synonym for CD71, TFR1, TR, T9, p90 and IMD46. Examples of TfR are human TfR, containing the amino acid sequence presented in the GenBank database under the accession number NP_003225 (http://www.ncbi.nlm.nih.gov/) or the sequence SEQ ID NO: 6; simian TfR containing the amino acid sequence represented by SEQ ID NO: 8, and the like. Also included are polypeptides consisting of an amino acid sequence that is the product of a deletion, substitution, or insertion of one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 6, sequence accession number NP_003225 in the GenBank database or SEQ ID NO: 8, and having the TfR function.

The transferrin receptor of the present invention also includes polypeptides containing an amino acid sequence having an overall 70% or more, preferably 80% or more, and more preferably 90% or more homology with the amino acid sequence represented by SEQ ID NO: 6, sequence accession number NP_003225 in the GenBank database or SEQ ID NO: 8, and most preferably, polypeptides consisting of an amino acid sequence that is 95%, 96%, 97%, 98%, 99% or more homologous to the specified sequence and has TfR function.

A polypeptide whose amino acid sequence is the product of a deletion, substitution, or insertion of one or more amino acid residues in the sequence represented by SEQ ID NO: 6, GenBank accession number NP_003225 or SEQ ID NO: 8, can be obtained, for example, by introducing site-specific mutations into DNA encoding SEQ ID NO: 6, sequence with registration number NP_003225 in the GenBank database or SEQ ID NO: 8, by site-specific mutagenesis [Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proceeding of the National Academy of Sciences in USA, 82, 488 (1985)] or the like. The number of amino acid residues to be deleted, replaced or inserted is not particularly limited, but is preferably from one to several tens, such as 1-20, more preferably from one to ten, such as 1-5.

Examples of the gene encoding TfR are, for example, the nucleotide sequence encoding human TfR represented by SEQ ID NO: 5 or sequence accession number NM_003234 in the GenBank database; the nucleotide sequence encoding simian TfR represented by SEQ ID NO: 7, and other similar genes. The genes encoding the transferrin receptor of the present invention also include genes consisting of a nucleotide sequence that is the product of a deletion, substitution or insertion of one or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 5, sequence accession number NM_003234 GenBank or SEQ ID NO: 7, and containing DNA encoding a polypeptide having TfR function; genes consisting of a nucleotide sequence that is preferably 60% or more, more preferably 80% or more, even more preferably 95% or more homologous to the nucleotide sequence represented by SEQ ID NO: 5, sequence accession number NM_003234 GenBank or SEQ ID NO: 7, and containing DNA encoding a polypeptide having TfR function; genes consisting of DNA that, under stringent conditions, hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5, sequence with GenBank accession number NM_003234 or SEQ ID NO: 7, and containing DNA encoding a polypeptide having TfR function.

DNA that hybridizes under stringent conditions in this case means, for example, hybridizing DNA obtained by colony hybridization, plaque hybridization, Southern blotting or DNA microarrays, or other methods in which DNA having the nucleotide sequence represented by SEQ ID NO: 5 or the sequence with registration number NM_003234. Specifically, this is, for example, DNA that is identified by washing a filter or slide at 65°C with a SSC solution at a concentration of (0.1-2)x (composition of a 1x SSC solution: 150 mmol/l sodium chloride and 15 mmol/l sodium citrate) after hybridization [Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), DNA Cloning 1: Core Techniques, A Practical Approach, 2nd edition, Oxford University (1995)] at 65°C in the presence of sodium chloride at a concentration of 0.7-1.0 mol/l using a filter or glass slide on which DNA is immobilized from colonies or plaques used for hybridization, or a polymerase chain reaction (PCR) product, or an oligodeoxyribinucleotide having the specified nucleotide sequence. An example of DNA capable of such hybridization is DNA that is preferably 60% or more, more preferably 80% or more, even more preferably 95% or more homologous to the nucleotide sequence represented by SEQ ID NO: 5 or sequence accession number NM_003234.

In eukaryotes, polymorphism in the nucleotide sequences of genes encoding proteins is often observed. According to this invention, the concept of “gene encoding the transferrin receptor” also includes polymorphic variants of the gene that are the product of small mutations (substitutions, deletions or insertions of one or more nucleotides) of the nucleotide sequence.

The numerical expression of sequence homology of this invention is calculated using homology search programs known to those skilled in the art (unless otherwise stated); in particular, for nucleotide sequences, the default parameters of the BLAST program are used [J. Mol. Biol., 215, 403 (1990)]; for amino acid sequences, the default parameters of the BLAST 2 program are used [Nucleic Acids Research, 25, 3389 (1997), Genome Research, 7, 649 (1997), http://www.ncbi.nlm.nih.gov/Education/BLASTinfo /information3.html] and similar tools.

As for the default parameters, the penalty for opening a gap (G) in the case of nucleotide sequences was 5, in the case of amino acid sequences 11; gap extension penalty (-E) in the case of nucleotide sequences 2, in the case of amino acid sequences 1; penalty for nucleotide mismatch (-q) -3; nucleotide match bonus (-r) 1; expected number of events (-e) 10; word length (-W) in the case of nucleotide sequences 11, in the case of amino acid sequences 3; parameter (-y) [exclusion (X) extensions in bits] in the case of BLASTN 20, in the case of other programs of the BLAST 7 family; parameter (-X) (X exclusion for alignment with missing bits) 15, parameter -Z (final exclusion X for alignment with missing bits) in the case of BLASTN 50, in the case of other programs of the BLAST 25 family (http://www. ncbi.nlm.nih.gov/blast/html/blastcgihelp.html).

A polypeptide consisting of a portion of the amino acid sequence of TfR can be prepared using methods known to those skilled in the art; for example, a portion of the DNA encoding the amino acid sequence corresponding to SEQ ID NO: 6, GenBank accession number NP_003225 or SEQ ID NO: 8, is excised, and cells transformed with the vector containing the resulting DNA are cultured. It is also possible to obtain, for example, a polypeptide having an amino acid sequence that is the product of a deletion, substitution, or insertion of one or more amino acid residues within a portion of the amino acid sequence represented by SEQ ID NO: 6, GenBank accession number NP_003225, or SEQ ID NO: 8 ; for this purpose, the same method is used as described above, starting from the polypeptide or DNA obtained by the method described above. In addition, a polypeptide consisting of a portion of the TfR amino acid sequence, or a polypeptide with an amino acid sequence that is the product of a deletion, substitution, or insertion of one or more amino acid residues in a portion of the TfR amino acid sequence, can be prepared by chemical synthesis using, for example, the fluorenylmethyloxycarbonyl method (Fmoc) or tert-butyloxycarbonyl (tBoc).

Using programs that allow you to predict transmembrane regions of a protein, namely SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM (version 2) (http://www.cbs.dtu .dk/services/TMHMM-2.0/), ExPASy Proteomics Server (http://Ca.expasy.org/) and other similar tools, you can determine the extracellular domain of the TfR of the present invention, for example, determine the corresponding region in the amino acid sequence of human TfR , represented by sequence accession number NP_003225 in the GenBank database. Specifically, it is part of the amino acid sequence represented by SEQ ID NO: 2, or sequence accession number NP_003225 in the GenBank database, from position 89 to position 760.

An example of a TfR function is the uptake of iron, which is vital for cell function and proliferation. When the iron-transferrin complex binds to TfR, it enters the cell by endocytosis. In the endosome, the pH decreases, and iron is released from the complex with transferrin and moves into the cytoplasm with the participation of the divalent metal ion transporter protein (DMT1), where it is used in cell proliferation or energy production. It is known that the complex of TfR and its receptor, from where iron is released, usually does not disintegrate after this and returns to the cell surface (Yamashiro D. J. et al., Cell, 37 789-800, 1984).

Examples of cells that express the transferrin receptor include cells from many normal tissues, including bone marrow and placenta; cancer cells of many different types, including colon, head and neck, brain, hematopoietic tissue, liver and esophagus, and many cancer cell lines, such as HT29, HSC-2, RAMOS, K562, HepG2, OE21, T .Tn, U-937, HuH-7 and HLE.

The term “cell surface antigen” of the present invention refers to antigens other than TfR, among antigens such as proteins and polypeptides expressed on the cell membrane of cancer cells or the like. The cell surface antigen of the present invention can be anything as long as the protein or polypeptide of which the antibody is an example can bind to it, but preferably it is a cell surface antigen that is internalized and/or degraded by binding to the protein or polypeptide, e.g. with antibody. Also, the cell surface antigen of the present invention is preferably a protein that is highly expressed in cancer cells or in those cell populations that are involved in a particular disease. Preferable examples of cell surface antigens of the present invention include EGFR, GPC3 and the like.

The designation EGFR is used herein synonymously with the designations ERBB, ERBB1, HER1, PIG61, MENA, NISBD2, SA7 and c-Erb-1.

An example of EGFR is, for example, human EGFR containing the amino acid sequence represented by GenBank accession number NP005219 or SEQ ID NO: 14; simian EGFR containing the amino acid sequence represented by the sequence accession number XP_005549616.1 in GenBank. Examples of EGFR also include polypeptides with an amino acid sequence that is the product of a deletion, substitution or insertion of one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 14 or sequences with GenBank accession numbers NP005219 and XP_005549616.1 and having EGFR function.

The EGFR of this invention also includes polypeptides having preferably 70% or more, more preferably 80% or more, even more preferably 90% or more homology to the amino acid sequence represented by SEQ ID NO: 14 or sequence accession numbers NP005219 and XP_005549616.1 in GenBank, and most preferably includes polypeptides consisting of an amino acid sequence having 95%, 96%, 97%, 98% and 99% or more homology with the specified amino acid sequence and having EGFR function.

A polypeptide whose amino acid sequence is the product of a deletion, substitution, or insertion of one or more amino acid residues in the sequence represented by SEQ ID NO: 14 or GenBank accession numbers NP005219 and XP_005549616.1 can be obtained, for example, by introducing site-specific mutations in DNA encoding the amino acid sequence represented by SEQ ID NO: 14 or sequences with GenBank accession numbers NP005219 and XP_005549616.1, by site-directed mutagenesis or the like. The number of amino acid residues to be deleted, replaced or inserted is not particularly limited, but is preferably from one to several tens, for example 1-20, more preferably from one to several, for example 1-5.

An example of a gene encoding the EGFR of the present invention is, for example, the human EGFR gene containing the nucleotide sequence represented by SEQ ID NO: 13 or the sequence registered in GenBank under the number NM_005228.

The EGFR-encoding gene of the present invention also includes genes consisting of a nucleotide sequence that is the product of a deletion, substitution or insertion of one or more nucleotides in the sequence represented by SEQ ID NO: 13 or the sequence registered in GenBank number NM_005228, and containing DNA , which encodes a polypeptide having EGFR function; genes consisting of a nucleotide sequence that is 60% or more, preferably 80% or more, more preferably 95% or more homologous to the nucleotide sequence represented by SEQ ID NO: 13 or the sequence registered in GenBank number NM_005228, and containing DNA , encoding a polypeptide having EGFR function; genes consisting of DNA that, under stringent conditions, hybridizes with DNA containing the nucleotide sequence represented by SEQ ID NO: 13 or the sequence registered in GenBank under the number NM_005228, and which contains DNA encoding a polypeptide having EGFR function, and similar genes.

Using programs that allow you to predict transmembrane regions of a protein, namely SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM (version 2) (http://www.cbs.dtu .dk/services/TMHMM-2.0/), ExPASy Proteomics Server (http://Ca.expasy.org/) and other similar tools, the extracellular domain of the EGFR of the present invention is established, for example the corresponding region in the amino acid sequence of human EGFR represented by the sequence , registered in the GenBank database under number NP005219. Specifically, it is part of the amino acid sequence represented by SEQ ID NO: 10 or the sequence registered in the GenBank database under the number NP005219, from the 25th position to the 645th position.

The function of EGFR is to bind EGF, after which a dimer is formed, and as a result, the Ras/Raf/MAPK, PI3K/Akt or Jak/STAT signaling pathways are triggered, promoting cell proliferation, cell survival, cell migration and cell invasion. It is known that after binding to epidermal growth factor, its receptor is internalized and, having reached lysosomes as a result of endocytosis, is cleaved there (Ebner R et al., Cell Regul. 2 599-612, 1991).

Examples of cells in which EGFR is expressed include epithelial cells of normal tissues, including skin, colon, and lung, as well as epithelial cancer cells, including colon, head and neck, lung, and esophageal tumors, and cells of cancer cell lines eg HT29, HSC-2, NCI-H1975, OE21, T.Tn, and A431.

In this document, the designation GPC3 is used as a synonym for the designations DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS and SGBS1.

GPC3 functions by binding to protein associated with the Wnt or Frizzled signaling pathway, and as a result is involved in cell division or proliferation of cancer cells, such as liver cancer.

An example of GPC3 is human GPC3, which contains the amino acid sequence registered in the GenBank database under the number P51654.

Using programs that allow you to predict the transmembrane region of a protein, namely SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM (version 2) (http://www.cbs.dtu .dk/services/TMHMM-2.0/), ExPASy Proteomics Server (http://Ca.expasy.org/) and other similar tools, the extracellular domain of GPC3 according to the present invention is established, for example the corresponding region in the amino acid sequence of human GPC3 represented by the sequence , registered in the GenBank database under number P51654. Specifically, it is the amino acid sequence represented by the sequence registered in the GenBank database under the number P51654, from positions 1 to 559.

Examples of cells in which GPC3 is expressed include fetal liver cells, as well as cancer cells, such as liver cancer cells, and liver cancer cell lines, such as HepG2 and HuH-7

An antibody is a protein “derived from” a gene (referred to as an antibody gene) encoding all or part of the heavy chain variable region, heavy chain constant region, light chain variable region and light chain constant region that form the immunoglobulin molecule. For the purpose of this invention, the term antibody includes antibodies or antibody fragments of any class or subclass of immunoglobulins.

The term "heavy chain" (denoted H chain) refers to a polypeptide with a higher molecular weight than the other type of polypeptide of the two that form the immunoglobulin molecule. The heavy chain determines the class and subclass of the antibody. There are five classes of antibodies: IgA, IgD, IgE, IgG and IgM, respectively, according to five types of heavy chains α, δ, ε, γ and μ, differing in the amino acid sequence of the constant region. The term "light chain" (denoted L chain) refers to a polypeptide with a lower molecular weight than the other type of polypeptide of the two that make up the immunoglobulin molecule. In humans, there are two types of light chains - κ and λ.

The variable (V) region is usually called the region with high variability on the N-terminal side of the immunoglobulin molecule. Since the structure of the remaining part varies less, it is called constant (C). The corresponding variable regions of the heavy and light chains are associated with the formation of the antigen-binding domain, which determines the ability of the antibody to bind to the antigen.

In the heavy chain of human antibodies, part of the amino acid sequence from position 1 to position 117 is variable (the numbering of amino acid residues is indicated in the EU numbering system, consistent with the Cabot system (Kabat et al., Sequences of proteins of immunological interest, 199; 5- e edition), and the constant region is the region of the polypeptide chain below position 118. In the light chain of human antibodies, the variable region is the region from the 1st to the 107th position of the amino acid sequence (Cabot numbering of amino acid residues), and the constant region is the region of the polypeptide chain below position 108. The variable regions of the heavy and light chains are designated VH or VL, respectively

An antigen-binding domain is a domain that recognizes and binds to antigen; this term refers to domains that form a conformation that is complementary to an antigenic determinant (epitope). The antigen-binding domain provides a strong intermolecular interaction with the antigenic determinant. The antigen-binding domain of an antibody molecule is formed by the variable regions of the light and heavy chains; having at least three hypervariable regions that determine complementarity with the epitope (CDR regions). In human antibodies, VH and VL each have three CDR regions, which are designated CDR1, CDR2 and CDR3, counting from the N-terminus of the polypeptide chain.

In the constant region; the heavy and light chain constant regions are designated CH and CL, respectively. Heavy chain constant regions are classified as α chain, δ chain, ε chain, γ chain, and μ chain. The heavy chain constant region consists of the CH1, hinge, CH2, and CH3 domains (indicated from the N-terminus of the polypeptide chain), with the CH2 and CH3 domains together forming a region called the Fc. In turn, the constant regions of light chains are classified into Cλ chain and Cκ chain.

Monoclonal antibodies are antibodies secreted by antibody-producing cells that maintain monoclonality and recognize one specific epitope (antigenic determinant). All monoclonal antibody molecules have the same amino acid sequence (primary structure) and the same spatial structure. Polyclonal antibodies are a population of antibody molecules secreted by antibody-producing cells of different lineages. Oligoclonal antibodies are a population of antibody molecules in which different monoclonal antibodies are mixed.

An epitope is a structural domain of an antigenic molecule that is recognized and bound by an antibody. Examples of epitopes include a single amino acid sequence, a spatial structure formed by an amino acid sequence; an amino acid sequence to which a sugar-based chain is attached; a structure formed by an amino acid sequence to which is attached a sugar chain, and the like, each of which is recognized and bound by a monoclonal antibody.

Examples of monoclonal antibodies of the present invention include antibodies produced by hybridomas and genetically engineered recombinant antibodies produced by cells that are transformed with an expression vector including an antibody gene.

A hybridoma can be produced, for example, by obtaining an antigen, obtaining an antibody-producing cell having antigen specificity from an animal immunized with the antigen, and then fusing the antibody-producing cell with a myeloma cell. The desired monoclonal antibody can be obtained by culturing the hybridoma or injecting the hybridoma into an animal to convert the hybridoma into an ascites tumor, separating the culture solution or ascites, and then purifying it. As the animal for immunization with the antigen, any animal can be used as long as it can produce a hybridoma, but it is preferable to use a mouse, rat, hamster, rabbit or the like. In addition, a hybridoma can also be obtained by obtaining a cell having the ability to produce antibodies from such an immunized animal, immunizing this cell in vitro and then fusing this cell with a myeloma cell.

Examples of the genetically engineered recombinant antibody of the present invention include antibodies produced using recombinant DNA technology, such as recombinant mouse antibodies, recombinant rat antibodies, recombinant hamster antibodies, recombinant rabbit antibodies, human chimeric antibodies (also referred to herein as simply chimeric antibodies), humanized antibodies (also called CDR-grafted antibodies) and human antibodies. Depending on the animal species to be used as the target and the target, applicable heavy chain and light chain variable regions and constant regions derived from the given animal species can be determined for the genetically engineered recombinant antibody. For example, when the animal species to be used as a target is a human, a region derived from a human or a non-human animal such as a mouse may be adopted as the variable region, and a region derived from a human or a non-human animal such as a mouse may be adopted as the constant region and linker. , those obtained from humans.

A chimeric antibody refers to an antibody consisting of the VH and VL of a non-human animal (non-human animal) antibody and the CH and CL of a human antibody. As the non-human animal, any animal such as a mouse, rat, hamster or rabbit can be used as long as it can produce a hybridoma. A chimeric antibody can be produced by obtaining cDNAs encoding VH and VL from a hybridoma obtained from a non-human animal that produces a monoclonal antibody, by inserting each of the cDNAs into an expression vector for an animal cell having DNA encoding CH and CL of the human antibody , thereby constructing a chimeric antibody expression vector and then introducing the vector into an animal cell to induce expression.

A humanized antibody is an antibody in which CDRs from the VH and VL of a non-human animal antibody are grafted into place of the corresponding CDRs from the VH and VL of a human antibody. The region other than the CDR of VH and VL is called the frame region (hereinafter referred to as FR). A humanized antibody can be produced by constructing a cDNA encoding a VH amino acid sequence consisting of the CDR amino acid sequences from a VH of a non-human animal antibody and an FR from a VH of an arbitrary human antibody, and a cDNA encoding a VL amino acid sequence consisting of a CDR amino acid sequence from a VL a non-human animal antibody and the FR amino acid sequence from the VL of an arbitrary human antibody, inserting each of the cDNAs into an animal cell expression vector containing DNAs encoding the CH and CL of the human antibody, thereby constructing a humanized antibody expression vector, and then introducing the vector into animal cell to induce expression.

The term "human antibody" originally refers to antibodies naturally present in the human body, but it also includes antibodies obtained from phage libraries of human antibodies and from transgenic animals producing human antibodies, the production of which is made possible by recent advances in gene, cell or tissue engineering and the like.

To obtain antibodies naturally present in the human body, one can, for example, infect peripheral blood lymphocytes with EB virus or the like to immortalize the lymphocytes, and clone these lymphocytes to obtain a culture of lymphocytes producing the desired antibodies, which are isolated from the culture supernatant and purified.

A human antibody phage library is a library in which an antibody fragment, such as Fab or scFv fragments, is expressed on the surface of a phage by inserting a human B cell-derived antibody gene into the phage gene. It is possible to isolate a phage that expresses on the surface an antibody fragment having the desired antigen-binding activity from a library using the binding activity to the substrate on which the antigen is immobilized as an indicator. The antibody fragment can be further converted into a human antibody molecule consisting of two complete H chains and two complete L chains using genetic engineering.

A human antibody transgenic animal is an animal that has a human antibody gene incorporated into its cell. In particular, for example, a transgenic mouse producing human antibodies can be produced by introducing a human antibody gene into mouse ES cells, implanting the ES cell into an early mouse embryo, and then growing it from an individual embryo. A human antibody obtained from a transgenic animal producing human antibodies can be produced by producing a hybridoma using a conventional hybridoma production method, which is performed by using a non-human animal and culturing the hybridoma, thereby producing and accumulating the antibody in the culture supernatant.

The CH of the genetically engineered recombinant antibody can be any polypeptide chain belonging to a human immunoglobulin, but it is preferable to take the CH of human immunoglobulins G (hIgG). In addition, CH from any of the subclasses of the hIgG class, such as hIgG1, hIgG2, hIgG3 and hIgG4, can be used. The CL of a genetically engineered recombinant antibody can be any polypeptide chain belonging to human immunoglobulin; κ or λ class light chain CL may be used.

As used herein, a bispecific antibody is a polypeptide or protein that specifically binds to two different epitopes. Each of the antigen-binding domains of a bispecific antibody may bind to distinct epitopes of the same antigen or may bind to different antigens.

In accordance with the present invention, the binding of a polypeptide, antibody or fragment of a given antibody, or a bispecific fragment of a given antibody to any cell surface antigen, such as EGFR or GPC3 and TfR, can be confirmed using a method in which the binding affinity of the antibody of interest to a cell that expresses the antigen cell surface or TfR is confirmed using, for example, a known immunological detection method, preferably a method of staining cells with a fluorescent dye or the like. In addition, known immunological detection methods can also be used [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experimental Manual, Kodansha scientific books ( 1987)], etc. in combination.

In accordance with the present invention, the antigen binding domain that binds to a cell surface antigen such as EGFR or GPC3 or TfR can be any one as long as it specifically recognizes and binds to a cell surface antigen such as EGFR or GPC3 or TfR. For example, the domain may be in any form of a polypeptide, a protein molecule and a fragment thereof, a conjugate of a body with a small molecule or a natural product of a protein molecule, etc., which can be obtained by gene recombination, for example, an antibody, a ligand, a receptor, or a naturally occurring interacting molecule.

Also, the antigen-binding domain can be a recombinant binding protein that uses a known antigen-binding agent, for example, an antibody, ligand or receptor; and specific examples include a recombinant protein having CDR regions of an antibody binding to said antigen, an antibody variable region having such CDRs, a recombinant protein comprising an antibody variable region and a ligand binding domain binding to said antigen, and the like. Preferably, the antigen binding domain of the present invention is the variable region of an antibody.

Examples of a bispecific antibody or bispecific antibody fragment of the present invention are bispecific antibodies or bispecific antibody fragments having TfR internalization and/or degradation activities. The bispecific antibody or bispecific antibody fragment of the present invention is preferably a bispecific antibody or bispecific antibody fragment that does not have TfR internalization and/or degradation activity on cells that do not express cell surface antigens such as EGFR or GPC3, but exhibiting this activity only against cells that express a cell surface antigen such as EGFR or GPC3. Such bispecific antibodies or bispecific antibody fragments selectively exhibit TfR internalization and/or degradation activity against pathogenic cells, for example cancer cells, that express cell surface antigens such as EGFR or GPC3, and therefore such antibodies or antibody fragments preferable from the point of view of the fact that they do not cause side effects that accompany nonspecific internalization and/or degradation of TfR.

By activity related to the internalization and/or degradation of TfR, which is possessed by bispecific antibodies or bispecific fragments of antibodies according to this invention, is meant the activity that results in the inclusion of TfR in the processes of internalization and/or degradation of a cell surface antigen upon binding of the specified bispecific antibody or a bispecific antibody fragment with both a cell surface antigen, such as EGFR or GPC3, and TfR, which causes internalization and/or degradation of TfR.

The bispecific antibody or bispecific antibody fragment of the present invention is capable of binding to a cell surface antigen such as EGFR or GPC3 and TfR expressed on the same cell, or to a cell surface antigen such as EGFR or GPC3 and TfR , expressed on different cells, but preferably they bind to a cell surface antigen, for example EGFR or GPC3, and TfR expressed on the same cell.

The bispecific antibody or bispecific antibody fragment of the present invention is preferably one that causes the death of a cell in which a target cell surface antigen, such as EGFR or GPC3, is expressed, thereby inducing internalization and/or degradation of TfRα.

The TfR internalization and/or degradation activity possessed by a bispecific antibody or bispecific antibody fragment of the present invention is confirmed by, for example, determining the level of TfR protein on the surface or within cells in which TfR is expressed, for example OE21, T.Tn cells , TE-8, U-937, HepG2, HuH-7 or HLE; It is also possible to determine the number of viable cells, cell proliferation, cell viability, cell iron uptake, etc.

Thus, examples of bispecific antibodies or bispecific antibody fragments of the present invention are, specifically, bispecific antibodies or bispecific antibody fragments that cause degradation of TfR upon binding to both a cell surface antigen, such as EGFR or GPC3, and TfR, and similar to them.

The number of domains that bind to a specific antigen in one bispecific antibody molecule is its binding valency. For example, according to the present invention, if one bispecific antibody molecule has two EGFR-binding domains and two TfR-binding domains, then the bispecific antibody binds each of the EGFR and TfR bivalently.

Bispecific antibodies of this invention also include antibodies that have multiple antigen-binding domains that are connected through appropriate linkers, including immunoglobulin domains or fragments thereof.

The bispecific antibodies of the present invention can be produced by methods known in the art ([Nature Protocols, 9, 2450-2463 (2014), WO 1998/050431, WO 2001/7734, WO 2002/002773 and WO 2009/131239) or the like .

The structure of the bispecific antibody of the present invention is such that a polypeptide (herein also referred to as an N-terminal side polypeptide) having the ability to bind a cell surface antigen is connected directly or via a linker to the N-terminus of an IgG portion. which binds to TfR.

According to the present invention, the immunoglobulin domain includes a peptide whose amino acid sequence is similar to that of the immunoglobulin and consists of 100 amino acid residues, among which at least two cysteine residues are present as the smallest unit. According to the present invention, the immunoglobulin domain also includes a polypeptide containing multiple immunoglobulin domains as its smallest unit as described above. Examples of the immunoglobulin domain include immunoglobulin heavy chain VH, CH1, CH2 and CH3, and immunoglobulin light chain VL and CL, and the like.

The species of the immunoglobulin is not particularly limited, but preferably the immunoglobulin is of human origin. The constant region may be any of the subclasses IgD, IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgE, and the subclasses of the IgG and IgM classes are preferred as examples. In addition, the immunoglobulin light chain constant region subclass can be either κ or λ.

Also, the immunoglobulin domain may be present in proteins other than immunoglobulins; for example, immunoglobulin domains are found in members of the immunoglobulin protein superfamily, such as the major histocompatibility complex (MHC), CD1, B7, and T-cell receptor (TCR) antigens. For the bispecific antibodies of this invention, any immunoglobulin domain can be used.

In the case of human IgG, CH1 is the region with the amino acid sequence from the 118th position to the 215th position, indicated according to the EU index. Similarly, CH2 is the region with amino acid sequence 231 to 340, indicated by the EU index according to Kabat et al., and CH3 is the region with amino acid sequence 341 to 447, indicated by the index EU by Kabat et al. Between CH1 and CH2 there is an amino acid flexibility region, also called a hinge region (hereinafter referred to as “hinge”). The hinge region is the region with the amino acid sequence from position 216 to position 230, indicated according to the EU index according to Kabat et al.

CL refers to the region with the amino acid sequence between positions 108 and 214, designated by Kabat in the case of the human antibody type κ light chain, and to the region with the amino acid sequence between positions 108 and 215 in the case of the λ type light chain.

According to this invention, the formulation antibody type IgG, also refers to the IgG part, and refers to the part of the IgG structure included in the bispecific antibody of the present invention, or an IgG in which the Fc fragment is modified and has a heterotetramer structure resulting from the assembly of two heterodimers, each of which consists of one light chain and one heavy chain. The portion of the IgG that binds to the TfR according to the present invention is that portion of the IgG that recognizes the TfR, that is, the portion of the IgG has the function of specifically recognizing and binding the extracellular domain of the TfR.

The IgG heavy chain constant region can be of any subclass, for example IgG1, IgG2, IgG3 or IgG4. Also, part of the amino acid sequence of this region can be deleted, added, replaced or inserted. In addition, all or part of the amino acid sequence fragments composed of the CH1, hinge, CH2 and CH3 regions of the IgG heavy chain can be suitably combined and used. Further, the amino acid sequences of these regions can be used, for example, with partial deletions or with a change in the order of their arrangement. The subclass of the IgG part used is not particularly limited, but preference is given to IgG4 or a mutant IgG4 obtained by replacing the Ser residue at position 228 of the heavy chain constant region with a Pro residue and the Leu residue at position 235 of the heavy chain constant region with an Asp residue (hereinafter this variant designated IgG4PE), or a mutant IgG obtained by replacing the Ser residue at position 228 of the heavy chain constant region with a Pro residue, the Leu residue at position 235 with an Asp residue, and the Arg residue at position 409 with a Lys residue (hereinafter this option is designated IgG4PE R409K ).

For example, an IgG portion is preferred in which the heavy chain constant region (CH1-hinge-CH2-CH3; listed in order from the N-terminal side) consists of IgG4PE containing the amino acid sequence represented by SEQ ID NO: 86, or an IgG portion in which the CH consists of IgG4PE R409K containing the amino acid sequence represented by SEQ ID NO: 84.

The two variable regions comprising the IgG portion of the invention preferably recognize the same antigen. Also, these regions preferably have the same structure and the same amino acid sequences.

In this invention, an N-terminal side polypeptide that binds a cell surface antigen is a portion of the bispecific antibody structure of the present invention, namely, a polypeptide located on the N-terminal side of the heavy or light chain of an IgG portion and that specifically recognizes and binds the extracellular region of each cell surface antigen. .

The bispecific antibody of the present invention has N-terminal side polypeptides located at the N-terminus of the heavy or light chain in both heterodimers constituting part of the IgG, or an N-terminal side polypeptide only at the N-terminus of one of the heterodimers; however, it is preferred that in said bispecific antibody, the N-terminal side polypeptides are present one at the N-terminus of the heavy or light chain in both heterodimers. In a bispecific antibody, when the N-terminal side polypeptides are present one at the N-terminus of the heavy or light chain in both heterodimers, the two polypeptides may be the same or different, but preferably they are the same.

The N-terminal side polypeptide of the present invention may be a single polypeptide chain or a multimer consisting of several polypeptide chains, provided that it has the ability to bind a cell surface antigen. The N-terminal side polypeptide may be antigen-binding fragments of an antibody to a cell surface antigen, namely Fab and Fab' fragments, scFv, dsFv and VHH, with Fab or VHH being preferred. Also, a ligand or receptor molecule specific for a cell surface antigen can be used as the N-terminal polypeptide of the present invention.

Antibodies to cell surface antigens of the present invention are monoclonal antibodies that specifically recognize and bind the extracellular domain of the corresponding cell surface antigen. For example, antibodies to EGFR and GPC3 are monoclonal antibodies that specifically recognize and bind the extracellular domain of EGFR and GPC3, respectively.

The linker of the present invention can be anything as long as it is a molecular structure through which the IgG portion and the N-terminal side polypeptide are connected, but preferably it is a peptide chain. Examples of such linkers are, for example, the amino acid sequences ES, ESKYG, ESKYGPP, GGGGS, or a sequence consisting of GGGGS repeats, or a sequence consisting of an amino acid sequence of a hinge region and a constant region of a CH1 domain, or a part thereof, or the like of antibodies, or similar sequences.

When in the bispecific antibody of the present invention, the N-terminal side polypeptide is a Fab of an antibody to a cell surface antigen, the light chain of the Fab and the light chain of the IgG part may be the same or different, but preferably they are the same. Here, said light chain may be of λ or κ type, but preferably κ.

Antibodies or antibody fragments that have an amino acid sequence that is the product of one or more substitutions, additions, deletions or insertions of amino acid residues in the amino acid sequences of a bispecific antibody or bispecific antibody fragment of this invention, and which have the activity of that antibody or fragment of that antibody are also included in scope of this invention.

The number of amino acid residues deleted, replaced, inserted and/or added may be one or more and is not particularly limited; is that number of deletions, substitutions, insertions, and additions of amino acid residues that can be accomplished using techniques well known in the art, such as site-directed mutagenesis, as described in Molecular Cloning, The Second Edition, Cold Spring Harbor Laboratory Press (1989 ), Current Protocols in Molecular Biology, John Willy & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc. For example. this amount can be from 1 to several tens, preferably 1-20, more preferably 1-10 and even more preferably 1-5.

The above description that one or more amino acid residues in the amino acid sequence of the bispecific antibody of the present invention can be deleted, replaced, inserted or added means the following. This description means that a deletion, substitution, insertion or addition of one or more amino acid residues has occurred at any one or more places in the same sequence. Further, such deletions, substitutions, insertions or additions of amino acid residues sometimes occur simultaneously, and the amino acid residues replaced, added or inserted may or may not be naturally occurring in naturally occurring proteins.

Examples of naturally occurring amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, 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, L-cysteine, etc.

Preferred examples of interchangeable amino acid residues are provided hereinafter. Amino acid residues belonging to the same group listed below are interchangeable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, O-methylserine, tert-butylglycine, tert-butylalanine and cyclohexylalanine.

Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid and 2-aminosuberic acid.

Group C: asparagine and glutamine.

Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid and 2,3-diaminopropionic acid.

Group E: proline, 3-hydroxyproline and 4-hydroxyproline.

Group F: serine, threonine and homoserine.

Group G: phenylalanine and tyrosine.

The bispecific antibody and the bispecific antibody fragment of this invention also include an antibody containing any amino acid residues that have undergone post-translational modification. Examples of post-translational modification include deletion of a Lys residue at the C-terminus of the H chain (Lys cleavage), replacement of a glutamine residue at the N-terminus of a polypeptide chain with pyroglutamate (pyroGlu), etc. [Beck et al, Analytical Chemistry, 85, 715-736 (2013 )].

Specific examples of the bispecific antibodies of the present invention include any bispecific antibody selected from the group consisting of the following (1) to (4) and the like:

(1) a bispecific antibody comprising a Fab that contains a variable region of an antibody to a cell surface antigen and a TfR binding portion of an IgG, the Fab being directly linked to the N-terminus of the heavy chain of the IgG portion;

(2) a bispecific antibody comprising a Fab that contains a VH having CDRs 1-3 of VH, and a VL having CDRs 1-3 of VH, wherein said VH and VL are derived from an antibody to a cell surface antigen, and an IgG portion that binds with TfR, and the Fab is directly connected to the N-terminus of the heavy chain of the IgG part;

(3) a bispecific antibody comprising a Fab that contains VH and VL antibodies to a cell surface antigen, and a TfR binding portion of an IgG, the Fab being directly linked to the N-terminus of the heavy chain of the IgG portion; And

(4) a bispecific antibody comprising a VHH that binds to a cell surface antigen and a TfR-binding portion of an IgG, wherein the VHH is directly linked to the N-terminus of the heavy chain of the IgG portion;

In the bispecific antibodies described in paragraphs (1) to (3) above, the VL in the TfR binding portion of the IgG and the Fab VL may be the same or different, but preferably they are the same.

An example of the TfR binding portion of the IgG of the present invention is the TfR binding portion of the IgG comprising VL comprising CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NOS: 17, 18 and 19, respectively, and VH containing CDR 1-3, examples of which are given below in paragraphs 1) - 3).

1) CDR1 containing the amino acid sequence represented by SEQ ID NO: 32, or the product of modification of the amino acid sequence represented by SEQ ID NO: 32 by at least one substitution selected from replacing tyrosine at position 2 with alanine, or phenylalanine and substituting threonine at position 3 to alanine or glycine.

2) CDR2 containing the amino acid sequence represented by SEQ ID NO: 33, or the product of modification of the amino acid sequence represented by SEQ ID NO: 33, by at least one substitution selected from the substitution of valine at position 1 with alanine, isoleucine at position 2 with alanine or leucine, valine at position 7 on glutamic acid, aspartic acid at position 10 on alanine, and aspartic acid at position 13 on proline.

3) CDR3 containing the amino acid sequence represented by SEQ ID NO: 34, or the product of modification of the amino acid sequence represented by SEQ ID NO: 34, by at least one substitution selected from the substitution of glutamine at position 3 with alanine or aspartic acid, proline at position 4 to a randomly selected natural amino acid, tryptophan at position 5 to alanine, phenylalanine, histidine or tyrosine, tyrosine at position 7 to alanine or phenylalanine, and valine at position 13 to leucine. When replacing the proline at position 4 with a randomly selected natural amino acid, examples of this latter are alanine, tyrosine, serine, aspartic acid, glutamine, glutamic acid, threonine, arginine, glycine, lysine, methionine, valine, leucine, isoleucine, tryptophan, phenylalanine or histidine .

The TfR binding portion of the IgG of the present invention also includes a TfR binding portion of the IgG comprising the amino acid sequences CDR 1-3 VH, and VL having 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% or at least 99% homology with the corresponding amino acid sequences CDR 1-3 VH represented by SEQ ID NOS: 32, 33 and 34, respectively, and the amino acid sequences CDR 1-3 VL represented by SEQ ID NOS: 17 - 19, respectively.

The TfR binding portion of the IgG of this invention also includes the antibodies described in (ai) or (aii) below.

(ai) A TfR-binding portion of an IgG that competes for binding to TfR with an anti-TfR antibody comprising VL with CDR regions 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively, and VH with CDR regions 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NOS: 32, 33, and 34, respectively; or

(aii) A portion of an IgG that binds to TfR and interacts with the same epitope as the anti-TfR antibody, comprising VL with CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively, and VH with CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NOS: 32, 33, and 34, respectively.

The TfR binding portion of the IgG of this invention also includes the antibodies described in (bi) or (bii) below:

(bi) an IgG portion that competes for binding to TfR with an anti-TfR antibody comprising VL with CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively, and VH with CDRs 1, 2 and 3, containing the amino acid sequences represented by SEQ ID NO: 42, 43 and 44, respectively; or

(bii) a portion of an IgG that binds to TfR by interacting with the same epitope as the anti-TfR antibody, comprising VL with CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively, and VH with CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 42, 43 and 44, respectively.

The TfR binding portion of the IgG of this invention also includes the antibodies described in (ci) or (cii) below:

(ci) a portion of an IgG competing for binding to TfR with an anti-TfR antibody comprising VL of CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively, and VH of CDRs 1, 2 and 3, containing the amino acid sequences represented by SEQ ID NO: 47, 48 and 49, respectively; or

(cii) a portion of an IgG that binds to TfR by interacting with the same epitope as the anti-TfR antibody, comprising VL with CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively, and VH with CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 47, 48 and 49, respectively.

Another example of the TfR-binding IgG portion of the present invention is the TfR-binding IgG portion comprising a VL comprising the amino acid sequence represented by SEQ ID NO: 16 and a VH comprising the amino acid sequence represented by SEQ ID NO: 26, 31 , 36, 41, 46 or 106.

One example of the TfR-binding IgG portion of the present invention is the TfR-binding IgG portion comprising a VL with CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 17-19, respectively, and a VH selected from the following :

(a) VH, comprising CDRs 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 33 and 34, respectively, and CDR1 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 32 by replacing the tyrosine at position 2 for alanine or phenylalanine;

(b) VH, comprising CDRs 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 33 and 34, respectively, and CDR1 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 32 by replacing threonine at position 3 for alanine or glycine;

(c) VH comprising CDRs 1 and 3 containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 33 by replacing valine at position 1 for alanine;

(d) VH comprising CDRs 1 and 3 containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 33 by replacing isoleucine at position 2 for alanine or leucine;

(e) VH, comprising CDRs 1 and 3 containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 33 by replacing valine at position 7 for glutamic acid;

(f) VH, comprising CDRs 1 and 3 containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 33 by replacing aspartic acid in position 10 to alanine;

(g) VH comprising CDRs 1 and 3 containing the amino acid sequences represented by SEQ ID NO: 32 and 34, respectively, and CDR2 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 33 by replacing aspartic acid in position 13 on proline;

(h) VH, comprising CDRs 1 and 2 containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 34 by replacing glutamine at position 3 for alanine or aspartic acid;

(i) VH, comprising CDRs 1 and 2 containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 34 by replacing a proline at position 4 per randomly selected amino acid;

(j) VH, comprising CDRs 1 and 2 containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 34 by replacing a proline at position 4 for alanine, tyrosine, serine, aspartic acid, glutamine, glutamic acid, threonine, arginine, glycine, lysine, methionine, valine, leucine, isoleucine, tryptophan, phenylalanine or histidine;

(k) VH, comprising CDRs 1 and 2 containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 34 by replacing tryptophan at position 5 for alanine, phenylalanine, histidine or tyrosine;

(l) VH, comprising CDRs 1 and 2 containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 34 by replacing the tyrosine at position 7 for alanine or phenylalanine; And

(m) VH, comprising CDRs 1 and 2 containing the amino acid sequences represented by SEQ ID NO: 32 and 33, respectively, and CDR3 containing the amino acid sequence being the product of modification of the amino acid sequence represented by SEQ ID NO: 34 by replacing valine with leucine .

One example of the TfR-binding portion of the IgG of the present invention is the TfR-binding portion of the IgG, comprising a VL comprising the amino acid sequence represented by SEQ ID NO: 16 and a VH containing an amino acid sequence resulting from a modification of the amino acid sequence represented by SEQ ID NO: 31, by at least one substitution selected from Y32A, Y32F, T33A, T33G, L45A, V48A, V50A, I51A, I51L, V55E, D58A, D61P, Q97A, Q97D, W99A, W99F, W99H, W99Y , Y100AA, Y100AF, V102L and P98 per randomly selected amino acid, as determined by the EU index as Kabat et al. (hereinafter EU index).

One example of the TfR-binding portion of an IgG of the present invention is a TfR-binding portion of an IgG comprising a VL containing the amino acid sequence represented by SEQ ID NO: 16 and a VH containing an amino acid sequence resulting from a modification of the amino acid sequence represented by SEQ ID NO: 31, by at least one substitution selected from Y32A, Y32F, T33A, T33G, L45A, V48A, V50A, I51A, I51L, V55E, D58A, D61P, Q97A, Q97D, W99A, W99F, W99H, W99Y , Y100AA, Y100AF, V102L, P98A, P98Y, P98S, P98D, P98Q, P98E, P98T, P98R, P98G, P98K, P98M, P98V, P98L, P98I, P98W, P98F and P98H, as defined by the EU index.

One example of the TfR-binding portion of the IgG of the present invention is the TfR-binding portion of the IgG that recognizes at least one amino acid residue selected from Asp at position 352, Ser at position 355, Asp at position 356, and Lys at position 358, and at least one amino acid residue selected from the residues Met at position 365, Val at position 366, and Glu at position 369 of the TfR amino acid sequence represented by SEQ ID NO: 6. In one embodiment of the present invention, the portion of the IgG that binds with TfR is, for example, a portion of an IgG that binds to TfR and recognizes at least two amino acid residues selected from the residues Asp at position 352, Ser at position 355, Asp at position 356 and Lys at position 358, and at least two amino acid residues selected from Met at position 365, Val at position 366 and Glu at position 369; the portion of an IgG that binds to TfR and recognizes at least three amino acid residues selected from Asp at position 352, Ser at position 355, Asp at position 356 and Lys at position 358, and all of the following amino acid residues: Met at position 365, Val at position 366 and Glu at position 369; and a portion of the IgG that binds to TfR and recognizes all of the following amino acid residues: Asp at position 352, Ser at position 355, Asp at position 356, and Lys at position 358, and all of the following amino acid residues: Met at position 365, Val at position 366 and Glu at position 369.

One example of an anti-EGFR antibody of the present invention is an anti-EGFR antibody having a VL comprising CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 17, 18 and 19, respectively, and any of the variable regions heavy chain (VH) selected from those given below in paragraphs (2a) to (2b).

(2a) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 60, 61 and 62, respectively; And

(2b) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 65, 66 and 67, respectively.

The anti-EGFR antibody of the present invention also includes antibodies that contain at least 35%, at least 40%, at least 45%, at least 50% CDR 1-3 VH and VL amino acid sequences. by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the corresponding amino acid sequences CDR 1 -3 VH, described in paragraphs (2a) and (2b) above, and the amino acid sequences CDR 1 - 3 VL represented by SEQ ID NO: 17 - 19, respectively.

The anti-EGFR antibody of the present invention also includes the antibodies described in (i) or (ii) below:

(i) an antibody that binds EGFR competitively with an antibody to EGFR, which has a VL comprising CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NOs: 17, 18 and 19, respectively, and a VH described by any of the above paragraphs (2a) - (2b); or

(ii) an antibody that binds the same epitope as an anti-EGFR antibody, having a VL comprising CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NOs: 17, 18 and 19, respectively, and a VH described by any from points (2a) - (2b) above.

Another example of an anti-EGFR antibody of the present invention is a bispecific antibody selected from the antibodies described in (a) to (e) below:

(a) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 59 or SEQ ID NO: 64, and a VL containing the amino acid sequence represented by SEQ ID NO: 16;

(b) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 110, and a VL chain containing the amino acid sequence represented by SEQ ID NO: 111;

(c) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 112, and a VL containing the amino acid sequence represented by SEQ ID NO: 113;

(d) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 114, and a VL containing the amino acid sequence represented by SEQ ID NO: 115; And

(e) an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 116 and a VL containing the amino acid sequence represented by SEQ ID NO: 117.

In one embodiment of the present invention, an example of a bispecific antibody is an antibody that includes an N-terminal side polypeptide and a TfR-binding portion of an IgG, and in which the N-terminal side polypeptide is a Fab fragment, wherein the C-terminus of the VH-CH1 region of the Fab is connected directly or via a linker to the N-terminus of the TfR-binding portion of the IgG.

Also in one embodiment of the present invention, an example of a bispecific antibody is an antibody that includes an N-terminal side polypeptide and a TfR binding portion of an IgG, and wherein the N-terminal side polypeptide is a Fab fragment, wherein the C terminus of the VL-CL region of the Fab is joined directly or via a linker to the N-terminus of the heavy chain of the TfR-binding portion of the IgG.

In one embodiment of the present invention, an example of a bispecific antibody is an antibody that includes a TfR binding moiety of an IgG and an N-terminal EGFR binding polypeptide.

More specific examples of the bispecific antibodies of this invention include the bispecific antibodies described in paragraphs (i) to (iv) below.

(i) A bispecific antibody comprising an anti-EGFR Fab antibody having a VL comprising CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NOS: 17, 18 and 19, respectively, and a VH comprising CDRs 1, 2 and 3, containing the amino acid sequences represented by SEQ ID NO: 60, 61 and 62, respectively, and comprising the TfR binding portion of an IgG in which there are VLs, including CDRs 1, 2 and 3, containing the amino acid sequences represented by SEQ ID NOS: 17, 18 and 19, respectively, and any VH selected from those described in (a) to (f) below, in which the Fab is connected directly or via a linker to the N-terminus of the heavy chain of the TfR binding portion of the IgG:

(a) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 22, 23 and 24, respectively;

(b) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 27, 28, and 29, respectively;

(c) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 32, 33, and 34, respectively;

(d) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 37, 38 and 39, respectively;

(e) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 42, 43 and 44, respectively; And

(f) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 47, 48 and 49, respectively.

(ii) A bispecific antibody comprising an anti-EGFR Fab, having a VL comprising CDRs 1, 2 and 3 comprising the amino acid sequences represented by SEQ ID NOs: 17, 18 and 19, respectively, and a VH comprising CDRs 1, 2 and 3, containing the amino acid sequences represented by SEQ ID NOS: 65, 66 and 67, respectively, and comprising a TfR binding portion of an IgG that contains a VL comprising CDRs 1, 2 and 3, containing the amino acid sequences represented by SEQ ID NOS: 17, 18 and 19, respectively, and any VH selected from those described in (a)-(f) below, wherein said Fab is connected directly or via a linker to the N-terminus of the heavy chain of the TfR binding portion of the Ig:

(a) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 22, 23 and 24, respectively;

(b) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 27, 28 and 29, respectively;

(c) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 32, 33 and 34, respectively;

(d) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 37, 38 and 39, respectively;

(e) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 42, 43 and 44, respectively; And

(f) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 47, 48 and 49, respectively.

(iii) A bispecific antibody comprising an anti-EGFR Fab, having a VL comprising CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NOs: 17, 18 and 19, respectively, and a VH comprising CDRs 1, 2 and 3, containing the amino acid sequences represented by SEQ ID NOS: 70, 71 and 72, respectively, and comprising the TfR binding portion of an IgG in which there are VLs, including CDRs 1, 2 and 3, containing the amino acid sequences represented by SEQ ID NOS: 17 , 18 and 19, respectively, and a VH selected from those described in (a) to (f) below, wherein said Fab is connected directly or via a linker to the N-terminus of the heavy chain of the TfR binding portion of the Ig:

(a) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 22, 23 and 24, respectively;

(b) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 27, 28 and 29, respectively;

(c) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 32, 33 and 34, respectively;

(d) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 37, 38 and 39, respectively;

(e) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 42, 43 and 44, respectively; And

(f) VH, including CDRs 1, 2 and 3 containing the amino acid sequences represented by SEQ ID NO: 47, 48 and 49, respectively.

(iv) A bispecific antibody comprising an anti-EGFR Fab, having a VL containing the amino acid sequence represented by SEQ ID NO: 16 and a VH containing the amino acid sequence represented by SEQ ID NO: 59 and including a TfR binding portion of an IgG , in which there are a VL comprising the amino acid sequence represented by SEQ ID NO: 16, and a VH comprising the amino acid sequence represented by SEQ ID NO: 21, 26, 31, 36, 41 or 46, in which the specified Fab is connected directly or through a linker to the N-terminus of the heavy chain of the TfR-binding portion of Ig.

(v) A bispecific antibody comprising an anti-EGFR Fab, having a VL containing the amino acid sequence represented by SEQ ID NO: 16 and a VH containing the amino acid sequence represented by SEQ ID NO: 64 and including a TfR binding portion of an IgG , in which there are a VL comprising the amino acid sequence represented by SEQ ID NO: 16, and a VH comprising the amino acid sequence represented by SEQ ID NO: 21, 26, 31, 36, 41 or 46, in which the specified Fab is connected directly or through a linker to the N-terminus of the heavy chain of the TfR-binding portion of Ig.

(vi) A bispecific antibody comprising an anti-EGFR Fab, having a VL containing the amino acid sequence represented by SEQ ID NO: 16 and a VH containing the amino acid sequence represented by SEQ ID NO: 69 and including a TfR binding portion of an IgG , in which there are a VL comprising the amino acid sequence represented by SEQ ID NO: 16, and a VH comprising the amino acid sequence represented by SEQ ID NO: 21, 26, 31, 36, 41 or 46, in which the specified Fab is connected directly or through a linker to the N-terminus of the heavy chain of the TfR-binding portion of Ig.

In the bispecific antibody described in (i) to (vi) above, the region connected directly or via a linker to the N-terminus of the heavy chain of the TfR binding portion of the IgG may be the VH-CH1 region or the VL-CL region of the Fab, but preferably VH-CH1.

An example of one embodiment of the present invention is a bispecific antibody in which the N-terminal side polypeptide is connected directly or via a linker to the N-terminus of the heavy chain of the TfR-binding portion of an IgG, and in which the constant region of the TfR-binding portion of the IgG is of a subtype IgG1, or IgG4, or their modifications. An example of a more preferred embodiment of the present invention is a bispecific antibody that includes IgG4PE or IgG4PE R409K in the heavy chain constant region of the TfR binding portion of the IgG, and in which the N-terminal side polypeptide is connected directly or via a linker to the N-terminus of the heavy chain of the IgG portion, binding to TfR.

An example of yet another embodiment of the present invention is a bispecific antibody that includes a TfR-binding portion of an IgG and an N-terminal side polypeptide that binds to the EGFR receptor, and wherein the N-terminal side polypeptide is a Fab fragment that is linked to the N-terminus the heavy chain of said IgG portion via a linker. An example of a more preferred embodiment of the present invention is a bispecific antibody wherein the linker has an amino acid sequence selected from the sequences ES, ESKYG, ESKYGPP, GGGGS or a GGGGS repeat sequence.

An example of a bispecific antibody of this invention is antibodies E08-TfR1071 or E12-TfR1071.

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

By effector activity according to this invention is meant the activity of antibody-dependent cellular cytotoxicity, which is mediated by the Fc region of the antibody molecule; examples of this activity include antibody-dependent cellular cytotoxicity (ADCC activity), complement-dependent cytotoxicity (CDC activity), antibody-dependent cellular phagocytosis (ADCP activity) involving phagocytic cells (eg, macrophages or dendritic cells), opsonization and etc.

According to this invention, ADCC and CDC activities are quantified using known analytical methods [Cancer Immunol. Immunother., 36, 373 (1993)].

ADCC activity is the activity that activates an immune cell (natural killer cell or similar) when an antibody that has bound an antigen on a target cell binds with its Fc region to the Fc receptor of the immune cell, resulting in damage to the target cell.

Fc receptor (FcR) is a receptor that binds to the Fc region of an antibody and induces various effector activities of the antibody. Each antibody subclass has its own FcR, and IgG, IgE, IgA, and IgM specifically bind to FcγR, FcγR, FcγR, and FcγR, respectively. In addition, FcγR has subtypes FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), and the subtypes have isoforms FcγRIA, FcγRIB, FcγRIC, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB, respectively. Different types of FcγR are present in 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.

CDC activity refers to the activity that activates a series of cascades (complement activation pathways) consisting of groups of complement-related proteins in the blood with the help of an antibody that binds to an antigen on a target cell to damage the target cell. In addition, a protein fragment formed during complement activation causes migration and activation of the immune cell. The cascade of CDC activity begins when C1q first binds to the Fc region and then binds to C1r and C1s, which are two serine proteases, resulting in the formation of the C1 complex.

The CDC activity or ADCC activity of a bispecific antibody or fragment thereof of the present invention against an antigen-expressing cell can be assessed by a known measurement method [Cancer Immunol. Immunother., 36, 373 (1993)].

As a method for controlling the effector activity of the bispecific antibody of the present invention, a method for controlling the amount of fucose (also called fucose core), which is α-1,6-linked to N-acetylglucosamine (GlcNAc) present at the reducing end of the N-linked sugar complex, is known. chain that binds to asparagine (Asn) at position 297 of the Fc region (constant region consisting of CH2 and CH3 domains) of an antibody (WO 2005/035586, WO 2002/31140 and WO 00/61739), a method of controlling effector activity by modifying the amino acid residue of the Fc region of an antibody (WO 00/42072), etc.

The ADCC activity of an antibody can be increased or decreased by adjusting the amount of fucose added to the bispecific antibody. For example, as a method of reducing the amount of fucose that binds to the N-linked complex sugar chain associated with the Fc of an antibody, expression of a bispecific antibody using a host cell deficient in the α1,6-fucosyltransferase gene can be used, which may result in an antibody with high ADCC was obtained. On the other hand, as a method of increasing the content of fucose that binds to the N-linked complex sugar chain associated with the Fc of a bispecific antibody, expression of the antibody using a host cell transfected with the α1,6-fucosyltransferase gene can be used, whereby obtain a bispecific antibody having low ADCC activity.

In addition, ADCC activity or CDC activity can be increased or decreased by modifying an amino acid residue in the Fc region of a bispecific antibody. For example, using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/0148165, the CDC activity of a bispecific antibody can be enhanced. In addition, by performing the amino acid modification described in US Pat. No. 6,737,056, US Pat. No. 7,297,775, US Pat. No. 7,317,091 and the like, ADCC activity or CDC activity can be increased or decreased.

In addition, a bispecific antibody in which effector activity is controlled can be obtained by combining the above methods.

The stability of the bispecific antibodies of this invention can be assessed by measuring the amount of aggregates (antibody oligomers) formed in a sample during purification and storage or under certain conditions. If, under the same conditions, the number of aggregates becomes smaller, then the stability of the antibody has improved. The number of aggregates can be determined by separating aggregated from non-aggregated antibodies using chromatographic methods, including gel filtration chromatography.

The yield of the bispecific antibody of the present invention can be assessed by measuring the amount of antibody produced by the antibody-producing cell in the culture solution. More specifically, productivity can be assessed by measuring the amount of antibody contained in the culture supernatant obtained by removing the producing cell from the culture solution using a suitable method such as an HPLC method or an ELISA method.

In the present invention, an antibody fragment is a protein that includes an antigen binding domain and has antigen binding activity towards an antigen. For example, Fab, Fab', F(ab')2, scFv, diabody, dsFv, VHH, peptide including CDR or the like.

Fab is an antibody fragment that has a molecular weight of about 50,000 and has antigen-binding activity, and in which approximately half of the H chain at the N-terminus and the entire L chain are linked by a disulfide bond (SS bond), obtained by treating an IgG antibody with the protease papain (with by cleavage at amino acid residue at position 224 in chain H). The H chain of Fab includes VH and CH1 and is designated VH-CH1. In this case, chain L of Fab includes VL and CL and is called VL-CL.

F(ab')2 is an antibody fragment that has a molecular weight of about 100,000, has antigen-binding activity, and is slightly larger than the molecule produced by binding a Fab through the S-S bond in the hinge region, produced by treating the IgG protease with pepsin (with cleavage at amino acid residue at position 234 in chain H).

Fab' is an antibody fragment which has a molecular weight of about 50,000 and has antigen-binding activity, and in which the S-S bond in the hinge region of the above F(ab')2 is cleaved.

ScFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (P) of 12 or more residues, and is an antibody fragment having antigen-binding activity.

A diabody is an antibody fragment in which scFvs having the same or different antigen-binding specificities form a dimer, and is an antibody fragment that has bivalent antigen-binding activity towards the same antigen or antigen-binding activity, each of which is specific for different antigens.

dsFv refers to a molecule obtained by linking polypeptides in which one amino acid residue in each of VH and VL is replaced by a cysteine residue through an S-S bond between the cysteine residues.

VHH (also called nanobody) is the heavy chain variable region in the VHH antibody and can bind to antigen without the presence of another polypeptide.

The VHH antibody is an antibody present in animals of the Camelidae family such as alpaca and elasmobranchs such as shark and does not include a light chain or CH1, and consists only of a heavy chain.

The CDR-including peptide is configured to contain at least one VH or VL CDR region. It is possible to obtain a peptide comprising multiple CDRs by attaching the CDRs directly or through a suitable peptide linker. To obtain a peptide including a CDR, DNA encoding the VH and VL CDRs of a bispecific antibody of the present invention is constructed, inserted into an expression vector for prokaryotic or eukaryotic cells, and introduced into the appropriate cells, resulting in expression. Also, the peptide including the CDR can be obtained by chemical synthesis using fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (tBoc).

In the present invention, the bispecific antibody fragment is essentially composed of a part of the structure of a bispecific antibody and is a bispecific antibody fragment having antigen binding activity against two types of antigens.

A fusion protein in which the Fc of a bispecific antibody of the present invention and an antibody fragment are linked, an Fc fusion protein (also called an immunoadhesin) in which an Fc and a naturally occurring ligand or receptor are linked, an Fc fusion protein in which multiple Fc regions are combined, etc. items bispecifically are also included in the present invention. In addition, the Fc region, which includes modification of an amino acid residue to enhance or eliminate the effector activity of the antibody, stabilize the antibody, and control the half-life in the blood, can also be used for the bispecific antibody of the present invention.

As the bispecific antibody or bispecific fragment of this antibody of the present invention, an antibody derivative in which a radioactive isotope, a low molecular weight drug, a high molecular weight drug, a protein, an antibody drug or the like included chemically or genetically engineered into a bispecific antibody or a bispecific fragment of this antibody according to the present invention.

The antibody derivative of the present invention can be obtained by coupling a radioisotope, low molecular weight drug, high molecular weight drug, immunostimulant, protein, antibody drug or the like to the N-terminal side or C-terminal side of the H chain or the L chain of a bispecific antibody or a bispecific fragment of the antibody of the present invention, a corresponding substituent or side chain of the antibody or antibody fragment, and a sugar chain or the like of the antibody or fragment of the antibody using a chemical method [Introduction to Antibody Engineering, Chijin Shokan Co ., Ltd. (1994)].

Derivatives of antibodies according to this invention can also be obtained by genetic engineering: DNA encoding a bispecific antibody or a bispecific fragment of an antibody according to this invention is ligated with DNA encoding the desired protein or drug antibody, and the resulting polynucleotide is inserted into an expression vector, which is introduced into suitable host cells and the desired product is expressed in them.

Examples of radioactive isotopes include ¹¹¹ In, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ⁶⁴ Cu, ⁹⁹ Tc, ⁷⁷ Lu, ²¹¹ At, etc. The radioactive isotope can be directly coupled to the antibody by a method using chloramine T or the like. It is also possible for a radioisotope chelating substance to be bound to the antibody. Examples of such chelating agents include 1-isothiocyanate-benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) and the like.

Examples of small molecule drugs include anticancer agents such as alkylating agents, nitrosoureas, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complexes, M phase inhibitors or kinase inhibitors [Clinical oncology, Japanese Journal of Cancer and Chemotherapy (1996)], anti-inflammatory agents such as steroidal compounds (hydrocortisone, prednisone, etc.), non-steroidal compounds such as aspirin or indomethacin, immunomodulators such as aurothiomalate or penicillamine, immunosuppressants such as cyclophosphamide or azathioprine, antihistamines such as chlorpheniramine maleate or clemastine [Inflammation and anti-inflammatory therapy, Ishiyaku Publishers, Inc. (1982)] and so on..

Examples of anticancer agents include amifostine (ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (Mustargen), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), epirubicin, gemcitabine (Gemzar) , daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, peplomycin, estramustine, paclitaxel (Taxol), docetaxel (Taxotere), aldesleukin, asparaginase, busulfan, carboplatin, oxaliplatin, nedaplatin , cladribine, camptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxyurea, idarubicin, 2-mercaptoethanesulfonic acid (mesnu), irinotecan (CPT-11), nogithecan, mitoxantrone, topotecan, leuprolide, megestrol , melphalan, mercaptopurine, hydroxyurea, plicamycin, mitotane, pegaspargase, pentostatin, pipobromane, streptozocin, tamoxifen, goserelin, leuprorelin, flutamide, teniposide, testolactone, thioguanine, thiotepa, uramustine, vinorelbine, chlorambucil, hydrocortisone, prednisolone, methylprednisolone , vindesine, nimustine , semustine, capecitabine, tomudex, azacitidine, tegafur/uracil (UFT), oxaloplatin, gefitinib (Iressa), imatinib (STI571), erlotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitors, vascular endothelial growth factor receptor (VEGFR) inhibitors, inhibitors fibroblast growth factor receptor (FGFR), EGFR inhibitors (eg, Tarceva), radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine, all-trans retinoic acid, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene (targretin), tamoxifen, dexamethasone, progestins, estrogens, anastrozole (arimidex), leuplin, aspirin, indomethacin, celecoxib, azathioprine, penicillamine, aurothiomalate, chlorpheniramine, maleate, chlorpheniramine, clemastine, tretinoin, bexarotene, arsenic, bortezomib , allopurinol, calicheamicin, ibritumomab, tiuxetan, targetin, ozogamine, clarithromycin, leucovorin, ketoconazole, aminoglutethimide, suramin, methotrexate or maytansinoid or their derivatives, etc.

Examples of methods used for binding a small molecule drug to a bispecific antibody or bispecific antibody fragment of the present invention include binding the drug to an amino group on an antibody molecule using glutaraldehyde, coupling the amino group of a drug to a carboxyl group on an antibody molecule using a water-soluble carbodiimide, etc.

Examples of high molecular weight drugs include polyethylene glycol (PEG), albumin, dextran, polyoxyethylene, styrene-maleic acid copolymers, polyvinylpyrrolidone, pyran copolymers, hydroxypropyl methacrylamide, and the like. As a result of the attachment of these high molecular weight substances to the bispecific antibody or antibody fragment of this invention, effects such as (1) increased stability when exposed to various chemical, physical or biological factors, (2) a significant extension of half-life in the blood or (3) disappearance of immunogenicity are expected or suppression of antibody formation [Bioconjugate pharmaceutical, Hirokawa-Shoten Ltd. (1993)].

Examples of methods for coupling PEG to a bispecific antibody of the present invention include interaction with PEGylation reagents and the like [Bioconjugate pharmaceutical, Hirokawa-Shoten Ltd. (1993)]. Examples of PEGylation reagents include modifying agents affecting the ε-amino group of lysine (JP A-S61-178926); modifying agents affecting the carboxyl group of aspartic or glutamic acid (JP-A-S56-23587); modifying agents affecting the guanidine group of arginine (JP-A-H2-117920), etc.

The immunostimulating agent of this invention may be a natural product called an immunoadjuvant; examples of such agents include substances that enhance the immune response, such as β(1→3)glucans (for example, lentinan or schizophyllan) or α-galactosylceramide (KRN7000), etc.

Examples of proteins include cytokines or growth factors that activate immune cells (eg, NK cells, macrophages or neutrophils), or toxic proteins, etc.

Examples of cytokines or growth factors include interferons (designated IFN-α, IFN-β, IFN-γ), interleukins (designated IL-2, IL-12, IL-15, IL-18, IL-21 and IL-23), colony-stimulating granulocyte factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), etc.

Examples of toxic proteins include ricin, diphtheria toxin, ONTAK, etc., as well as toxic proteins that have been mutated to control toxicity.

An antibody fused to another protein or drug antibody can be produced by ligase-linking a cDNA encoding that other protein to a cDNA encoding a bispecific antibody or a bispecific antibody fragment of the present invention, resulting in a DNA encoding the fusion antibody; the resulting DNA is inserted into an expression vector suitable for eukaryotic or prokaryotic cells, and the expression vector is introduced into the appropriate cells in which the fusion antibody is expressed.

When an antibody derivative of the present invention is used in a detection or quantitation method, or as a detection or quantitation reagent, or as a diagnostic agent, examples of a substance that is attached to the bispecific antibody or bispecific antibody fragment of the present invention include tag substances , which are used in immunological methods of detection or quantitative determination. Examples of tag substances include enzymes such as alkaline phosphatase, peroxidase or luciferase, luminescent substances such as acridine ester or triphenylimidazole (lofin), fluorescent substances such as fluorescein 5-isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC), Alexa family dyes (trade name), for example Alexa Fluor 488, or R-phycoerythrin (R-PE), etc.

The present invention includes bispecific antibodies and bispecific antibody fragments having cytotoxic activity, such as CDC or ADCC activity. The CDC or ADCC activity of bispecific antibodies or bispecific antibody fragments of the present invention directed to cells in which the antigen is expressed can be measured by known quantitation methods [Cancer Immunol. Immunother., 36, 373 (1993)].

This invention also relates to a composition containing as an active ingredient a bispecific antibody or bispecific fragment according to this invention that specifically recognizes and binds TfR and a cell surface antigen, for example EGFR, or a therapeutic agent containing a bispecific antibody or bispecific fragment according to this invention, for the treatment a disease associated with at least one of a TfR and a cell surface antigen, preferably one involving cells that express a TfR or a cell surface antigen, such as EGFR.

A disease associated with TfR or a cell surface antigen such as EGFR or both can be any one, provided that it is associated with TfR or a cell surface antigen such as EGFR or both, for example, malignant tumor, cancer, etc.

According to the present invention, examples of malignant tumors and cancers include colon cancer, colorectal cancer, lung cancer, breast cancer, glioma, malignant melanoma, thyroid cancer, renal cell carcinoma, leukemia, lymphoma, T-cell lymphoma, gastric cancer, pancreatic cancer glands, cervical cancer, uterine cancer, ovarian cancer, bile duct cancer, esophageal cancer, liver cancer, head and neck cancer, skin cancer, urinary tract cancer, bladder cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, cancer larynx, mesothelioma, pleural tumors, arrhenoblastoma, endometrial hyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi's sarcoma, angioma, cavernous hemangioma, angioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma stoma, osteogenic sarcoma, leiomyosarcoma , Wilms tumor, etc.

A therapeutic agent containing a bispecific antibody or bispecific antibody fragment of the present invention, or derivatives thereof, may contain only the bispecific antibody or bispecific antibody fragment of the present invention, or derivatives thereof, as an active ingredient, however, it is generally preferred that they be included a pharmaceutical preparation obtained by one of the methods known in the pharmaceutical industry by mixing with one or more pharmaceutically acceptable carriers.

As for the route of administration, the route of administration most effective for treatment is preferred; examples of which include oral or parenteral, such as oral, airway, intrarectal, subcutaneous, intramuscular and intravenous. Of these, the intravenous route of administration is preferred.

Examples of dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, patches, etc.

The dosage and frequency of administration of the drugs of this invention will vary depending on the desired therapeutic effect, the route of administration of the drug, the duration of treatment, the age and body weight of the individual or other factors; Typically, the daily dose for adults is 10 mcg/kg to 10 mg/kg.

This invention also relates to reagents for the immunological detection or quantitation of at least TfR or a cell surface antigen, for example, the EGFR receptor, or both; or agents for the diagnosis of diseases associated with at least TfR or a cell surface antigen, e.g. EGFR, or both, preferably diseases involving cells expressing at least TfR or a cell surface antigen, e.g. EGFR , or both of these substances, and these reagents and agents contain bispecific antibodies or bispecific antibody fragments according to this invention. In addition, this invention relates to a method for immunological detection or quantitation of at least TfR or a cell surface antigen, for example, EGFR, or both; to a method of treating diseases associated with at least TfR or a cell surface antigen, for example EGFR, or both, preferably diseases involving cells that express at least TfR or a cell surface antigen, for example EGFR, or both of these substances; or a method for diagnosing diseases associated with at least TfR or a cell surface antigen, for example EGFR, or both, preferably diseases involving cells that express at least TfR or a cell surface antigen, for example EGFR, or both of these substances; wherein said methods use bispecific antibodies or bispecific antibody fragments of the present invention.

An example of a method for detecting or quantifying at least a TfR or a cell surface antigen, such as EGFR, or both, may be any of the methods known in the art. For example, this could be an immunological detection or quantitation method.

An immunological detection or quantification method is one that uses a labeled antigen or antibody to detect or quantify an antibody or antigen. Examples of immunological detection or quantitation methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA, or ELISA), fluorescence immunoassay (FIA), luminescent immunoassay, Western blot, physicochemical methods, etc. .

By detecting or quantifying at least a TfR or a cell surface antigen (eg, EGFR), or both using bispecific antibodies or bispecific antibody fragments of the present invention, it is possible to diagnose diseases associated with at least a TfR or a cell surface antigen, e.g. , EGFR, or both, preferably diseases involving cells that express at least TfR or a cell surface antigen, such as EGFR, or both;

To identify cells that express at least TfR or a cell surface antigen, for example, EGFR, or both, known immunological methods can be used, for example, the immunoprecipitation method, the immunocytological staining method, the immunohistochemical staining method, the fluorescent antibody staining method, and others. similar methods. An example of a fluorescent antibody staining method is the FMAT 8100 HTS High Throughput Screening System (manufactured by Applied Biosystems, Inc.) or similar methods.

According to this invention, the biological sample for detection or quantification of at least TfR or a cell surface antigen, for example, EGFR, or both of these substances are tissue cells, blood, blood plasma, blood serum, pancreatic juice, urine, feces, tissue fluid, cultural environment, etc.; There is no limitation in the selection of the sample as long as it has the potential to contain cells expressing at least TfR or a cell surface antigen such as EGFR, or both.

A diagnostic agent comprising a bispecific antibody or bispecific antibody fragment of the invention, or derivatives thereof, may include a reagent for effecting the interaction of an antigen with an antibody or a reagent for detecting this interaction, according to the desired diagnostic method. Examples of reagents for interaction of antigen with antibody include buffer solutions, salts, etc.

Examples of reagents for detecting antigen-antibody interaction of the present invention include reagents used in conventional immunological detection or quantitation methods, such as labeled secondary antibodies that bind to a bispecific antibody, or a bispecific fragment of said antibody, or a derivative thereof, or a substrate corresponding label.

This document specifically describes a method for producing bispecific antibodies of the present invention, a method for determining the activity of bispecific antibodies or bispecific antibody fragments, a method for treating and a method for diagnosing diseases using bispecific antibodies or bispecific antibody fragments.

1. Method for producing monoclonal antibodies

The method for producing monoclonal antibodies of the present invention includes the following steps: (1) at least one of purifying an antigen to serve as an immunogen and obtaining cells overexpressing the antigen on the cell surface; (2) obtaining cells that produce antibodies by immunizing animals with this antigen, then taking their blood, determining the antibody titer in order to determine the moment of removal of the spleen or other material; (3) production of myeloma cells/myeloma; (4) fusion of antibody-producing cells with myeloma cells; (5) checking the resulting pool of hybridomas in order to identify those that produce the desired antibodies; (6) isolation (cloning) of monoclonal cells from a pool of hybridomas; (7) in some cases, cultivating hybridomas to produce monoclonal antibodies in large quantities or breeding animals with an implanted hybridoma; (8) studying the biological activity of monoclonal antibodies obtained in this way and their antigen specificity or studying properties as a labeling reagent, etc.

Hereinafter, the present document describes in detail, through the above steps, a method for producing TfR-binding monoclonal antibodies and cell surface antigen-binding monoclonal antibodies, such as EGFR, which are used to produce TfR-binding and cell surface antigen-binding bispecific antibodies, for example with EGFR, according to this invention. The method for producing antibodies according to this invention is not limited to the above: for example, the cells producing antibodies may not be spleen cells, but other cells, and instead of myeloma cells, other immortal cells.

(1) Antigen purification

Cells that express a cell surface antigen, such as EGFR, or TfR, can be obtained by introducing an expression vector containing cDNA encoding a full-length cell surface antigen, such as EGFR, or TfR, or part thereof, into E. coli, yeast, insect cells , animals and so on. Also, a cell surface antigen, such as EGFR, or TfR, or both, can be isolated from cultures of various human tumor cells or tissues where a cell surface antigen, such as EGFR, or TfR, or both, is highly expressed, purified, and used as an antigen. The cultured tumor cells or tissues themselves can also serve as antigens. In addition, a synthetic peptide with the amino acid sequence of a portion of a cell surface antigen molecule, such as EGFR, or TfR, can be produced by chemical synthesis (using Fmoc or tBoc) and also used as an antigen.

Cell surface antigen, such as EGFR, or TfR used in this invention can be obtained by methods described in Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989), or Current Protocols In Molecular Biology , John Wiley & Sons (1987-1997), or the like, for example, by expressing DNA encoding a cell surface antigen, such as EGFR, or TfR, in host cells as follows.

A full-length cDNA containing a region encoding a cell surface antigen, such as EGFR or TfRα, is inserted downstream of the promoter into a suitable expression vector. Instead of a full-length cDNA, a fragment of suitable length derived from the full-length cDNA and containing the region encoding the desired polypeptide can be used. The resulting recombinant vector is introduced into host cells suitable for its expression, and the transformed cells produce a cell surface antigen, such as EGFR, or TfR.

Any vector can be used as an expression vector as long as it is capable of autonomously replicating in selected host cells or can be incorporated into chromosomal DNA and contains a suitable promoter in a position suitable for transcription of DNA encoding a cell surface antigen, such as EGFR, or TfR.

The host cells can be any cells, for example microorganisms belonging to the genus Escherichia, such as E. coli, yeast, insect cells or animal cells, or other cells, provided that they can express the desired gene.

When using prokaryotic cells, such as E. coli, as host cells, a recombinant vector capable of autonomously replicating in prokaryotic cells and also comprising a promoter, a ribosome binding site, DNA containing a region encoding a cell surface antigen, such as EGFR, or TfR, and transcription termination sequence. A transcription terminator is not required in a recombinant vector, but if present, it is preferable that the termination sequence be located immediately after the structural gene. The recombinant vector may also contain a gene that controls a promoter.

The preferred recombinant vector is a plasmid in which the distance between the Shine-Dalgarno sequence (ribosome binding site) and the start codon is properly selected (for example, it is 6-18 nucleotides).

In a DNA sequence encoding a cell surface antigen, such as EGFR, or TfR, some nucleotides can be changed so that the codons become optimal for expression in a given host cell species, thereby increasing the production of a cell surface antigen, such as EGFR, or TfR, by those cells.

Any vector can serve as an expression vector, provided that it is capable of performing the desired function in the selected host cells; examples of such vectors include pBTrp2, pBTac1, pBTac2 (manufactured by Roche Diagnostics K.K.), pKK233-2 (manufactured by Pharmacia Corporation), pSE280 (manufactured by Invitrogen, Inc.), pGEMEX-1 (manufactured by Promega Corporation), pQE-8 (manufactured by QIAGEN, Inc.), pKYP10 (JP-A-S58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(-) (manufactured by Stratagene Corporation), pTrs30 [derived from E. coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [derived from E. coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [derived from E. coli IGHA2 (FERM BP-400), JP-A-S60-221091], pGKA2 [derived from E. coli IGKA2 (FERM BP-6798), JP-A -S60-221091], pTerm2 (US Patent Nos. 4,686,191, 4,939,094 or 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia Corporation), pET System (manufactured by Novagen, Inc.), pME18SFL3 (manufactured by Toyobo Co., Ltd.), etc.

The promoter can be any promoter capable of functioning in the selected host cells. Examples of such promoters include promoters from E. coli, promoters from bacteriophages or the like, such as the tryptophan (trp) operon (Ptrp) promoter, the lactose (lac) operon promoter, and the PL and PR promoters of bacteriophage λ or the bacteriophage T7 promoter. Moreover, examples of promoters used in the present invention include artificially constructed and modified promoters, such as a tandem promoter in which two Ptrp promoters are connected one after another; tac, lac-T7 or let I promoters, etc.

Examples of host cells used in this invention include 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, E. coli DH5α, etc.

Introduction of the recombinant vector into host cells can be accomplished by any method used to introduce DNA into cells; examples of such methods include methods using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

In the case of using animal cells as host cells for introducing the expression vector, such vector may be any suitable for animal cells, for example pcDNAI (manufactured by Invitrogen, Inc.), pcDM8 (manufactured by Funakoshi Co., Ltd.), pAGE107 [JP- A-H3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (JP-A-H2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (manufactured by Invitrogen, Inc.), pcDNA3.1 ( manufactured by Invitrogen, Inc.), pREP4 (manufactured by Invitrogen, Inc.), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, pKANTEX93 (WO 97/10354), etc.

The promoter of this invention can be any promoter capable of functioning in animal cells; examples of such promoters include the cytomegalovirus (CMV) immediate early (IE) gene promoter, the SV40 virus early gene promoter, the retroviral promoter, the metallothionein gene promoters, the heat shock protein gene promoters, the SRα promoter, and the Moloney murine leukemia virus promoter or enhancer. The human cytomegalovirus immediate early gene enhancer can also be used in conjunction with the promoter.

Examples of the host cells of the present invention include human Namalwa Burkitt lymphoma cells, African green monkey COS kidney cells, Chinese hamster ovary CHO cells, human leukemia cells HBT5637 (JP-A-S63-000299), etc.

Introduction of the recombinant vector into host cells can be accomplished by any method used to introduce DNA into cells; examples of such methods include electroporation [Cytotechnology, 3, 133 (1990)], the calcium phosphate method (JP-A-H2-227075), lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], etc.

A cell surface antigen, such as EGFR or TfR, can be produced by culturing microorganisms whose cells contain a recombinant vector comprising DNA encoding a cell surface antigen, such as EGFR or TfR, or transformants of the resulting animal cells, or other cells obtained as described above : in appropriate culture medium and conditions that allow the production of TfR, or a cell surface antigen, such as EGFR, or both, by a microorganism or transformant and their accumulation in the culture medium from which they are isolated. Cultivation of transformants is carried out using conventional methods used for culturing host cells.

In cases where the expression of a TfR or cell surface antigen, such as EGFR, is carried out in cells of eukaryotic origin, it is possible to obtain a TfR or a cell surface antigen, such as EGFR, with sugar moieties or sugar chains attached thereto.

When cultivating microorganisms transformed with a recombinant vector with an inducible promoter, an inducer is added to the culture medium if necessary. For example, in the case of cultivating microorganisms transformed with a recombinant vector with a lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is added to the culture medium, and in the case of cultivating microorganisms transformed with a recombinant vector with a trp promoter, indoleacrylic acid is added to the culture medium or similar connection.

Examples of culture media suitable for culturing transformants derived from animal cells include commonly used media such as RPMI 1640 [The Journal of the American Medical Association, 199, 519 (1967)], Eagle MEM [Science, 122, 501 (1952)], MEM Eagle's medium modified by Dulbecco [Virology, 8, 396 (1959)], medium 199 [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)], Iscove's modified Dulbecco's medium (IMDM), or said media supplemented with fetal bovine serum (FBS) or the like, or other similar media. Cultivation usually takes place for 1-7 days at a temperature of 30-40°C in an atmosphere containing 5% CO ₂ ; The pH of the culture medium is 6-8. If necessary, an antibiotic, such as kanamycin or penicillin, is added to the culture medium during the cultivation process.

To express genes encoding such cell surface antigens, EGFR or TfR, in addition to direct expression, the secretion production method, fusion protein expression, etc. can be used [Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989 )]. Examples of methods for producing cell surface antigens such as EGFR or TfR include production in host cells, secretion of these products from host cells, and production on the outer membrane of host cells; the most appropriate method is selected by changing the host cells or the structure of the cell surface antigen, such as EGFR or TfR, that is to be produced.

For example, it is possible to produce a protein fused to an antigen by producing DNA in which DNA encoding the Fc region of an antibody, DNA encoding glutathione S-transferase (GST), DNA encoding a FLAG tag, or DNA encoding a Histidine tag, or other nucleotide the sequence is ligated to DNA encoding the amino acid sequence of the extracellular domain, followed by expression and purification. Specific examples of this approach include the production of an Fc fusion protein in which the extracellular domain of a cell surface antigen (EGFR or TfR) is fused to the Fc region of a human IgG, and a fusion protein in which the extracellular domain of a cell surface antigen such as EGFR or TfR is fused to glutathione-S-transferase (GST).

When a cell surface antigen, such as EGFR or TfR, is produced in a host cell or on the outer membrane of a host cell, surface antigens such as EGFR or TfR can be actively secreted into the extracellular space using methods described in Paulson et al. J Biol. Chem., 264, 17619 (1989), Lowe et al. Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990) or in JP-A-H05-336963, in WO 94/23021 or similar methods. In addition, the amount of cell surface antigen produced, such as EGFR or TfR, can be increased using a desired gene amplification system using the dihydrofolate reductase gene or similar methods (see JP-A-H2-227075).

A host cell-produced cell surface antigen (eg, EGFR or TfR) is isolated and purified, for example, as follows.

If a cell surface antigen, such as EGFR or TfR, expressed in a host cell is in a dissolved state, then after culturing the host cells, they are collected by centrifugation, suspended in an aqueous buffer solution, homogenized using an ultrasonic homogenizer, a French press, a homogenizer Manton Gaulin Manufacturing Co., Inc., Dyno-Mill or other similar devices; the result is a cell-free extract solution. This solution is centrifuged and the desired protein is isolated from the resulting supernatant and purified using conventional methods for protein isolation and purification, such as solvent extraction, salting out with ammonium sulfate or other salt, desalting, precipitation using organic solvents, anion exchange chromatography on resins such as diethylaminoethyl (DEA). DEAE)-Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography on resins such as S-Sepharose FF (manufactured by Pharmacia Corporation), hydrophobic chromatography on resins such as butyl Sepharose or phenyl Sepharose, molecular sieve gel filtration , affinity chromatography, chromatofocusing, electrophoresis, including isoelectric focusing and other methods, individually or in combination.

If a host cell-expressed cell surface antigen, such as EGFR or TfR, forms insoluble particles, proceed as follows. Cells are collected and homogenized as described above and then centrifuged so that insoluble cell surface antigen particles, such as EGFR or TfR, are precipitated. The collected insoluble cell surface antigen particles, such as EGFR or TfR, are solubilized using a protein denaturing agent. The normal conformation of the cell surface antigen (EGFR or TfR) is restored by dilution or dialysis of the solubilized fraction. The cell surface antigen is then isolated and purified using the above methods.

If a cell surface antigen, such as EGFR or TfR, or a derivative thereof, such as a sugar-modified form, is secreted by a host cell into the extracellular space, then the cell surface antigen, such as EGFR or TfR, or a derivative thereof, such as a sugar-modified form, can be assembled from supernatant of the culture medium. Then the supernatant of the culture medium is processed by the methods described above, including centrifugation, and as a result a soluble fraction is obtained, from which the desired protein is isolated and purified by the methods described above.

Also, the cell surface antigen, such as EGFR or TfR, used in this invention can be obtained by chemical synthesis using the Fmoc or tBoc methods. Specifically, chemical synthesis is carried out using a peptide synthesizer manufactured by Advanced Chemtech, Inc., PerkinElmer, Inc., Pharmacia Corporation, Protein Technology Instrument, Inc., Synthecell-Vega Biomolecules Corporation, Perceptive, Inc., Shimadzu Corporation or other similar companies.

(2) Obtaining cells that produce antibodies.

Animals, such as mice, rats, hamsters, rabbits, cattle or alpacas, are immunized with the antigen obtained according to (1), and antibody-producing cells are isolated from the spleen, lymph nodes or peripheral blood of the immunized animal. Animals for immunization with the desired antigen can also serve, for example. transgenic mice that produce antibodies of human origin, described in the work of Tomizuka. et al., Proc. Natl. Acad. Sci. USA., 97, 722 (2000); mice with a conditional knockout of the gene encoding TfR or a cell surface antigen, such as EGFR, to enhance immunogenicity.

Immunization of animals is carried out by administering the antigen together with a suitable adjuvant, for example, Freund's complete adjuvant, aluminum hydroxide gel, Bordetella pertussis vaccine, etc. The administration of the immunogen to mice can be carried out by any method, for example, by subcutaneous injection, intravenous injection, intraperitoneal injection, intradermal injection, intramuscular injection , injections into the paw pad, etc.; Intraperitoneal, intravenous and footpad injections are preferred. In cases where the antigen is a partial peptide, the antigen is conjugate with a carrier protein, such as bovine serum albumin (BSA) or Fissurella apertura hemocyanin (KLH), and the conjugate is used as an immunogen.

The antigen is administered to animals 5-10 times with an interval of 1-2 weeks. 3-7 days after each administration, blood is taken from the immunized animal from the retro-orbital or sublingual venous plexus and the antibody titer in the collected blood serum is determined by enzyme-linked immunosorbent assay [Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] or another method. If the source of cells that produce antibodies, which are then used in fusion, are animals in which the titer of antibodies to the antigen used for immunization in the blood serum turned out to be sufficient, then the effect of subsequent processing is enhanced.

3-7 days after the last administration of the antigen, tissues containing antibody-producing cells, for example the spleen, are removed from immunized animals, and these cells are isolated. The cells that produce antibodies are lymphocytes - plasma cells and their precursors. These cells can be obtained from any site in the body, but typically the spleen, bone marrow, tonsils, peripheral blood, or combinations of these sources are used; Spleen cells are most commonly used. To isolate antibody-producing cells from the spleen, its tissue is crushed, a suspension is made, which is centrifuged, red blood cells are removed, and the resulting antibody-producing cells are suspended in a suitable medium.

(3) Receiving myeloma

To obtain myeloma, you can use mammalian cells, for example mice, rats, guinea pigs, hamsters, rabbits or humans, which are not capable of producing antibodies on their own; Typically, standard mouse cell lines are used, for example, 8-azaguanine-resistant myeloma cell lines P3-X63Ag8-U1 (P3-U1) derived from BALB/c mice [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)], P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)] or the like. Grow a subculture of such cells in a suitable medium, for example, 8-azaguanine medium [RPM1640 medium containing glutamine, 2-mercaptoethanol, gentamicin, fetal calf serum (FCS) and 8-azaguanine], Iscove's modified Dulbecco's medium (IMDM), Dulbecco's modified Eagle's medium (DMEM), etc. The above cell lines are subcultured in normal medium (for example, DMEM containing 10% FCS) for 3-4 days before fusion, so that on the day of fusion there are 2 x 10 ⁷ or more cells.

(4) Cell fusion

Antibody producing cells (APC) obtained in (2) and myeloma cells (MC) obtained in (3) were washed well with minimal essential medium (MEM) or PBS (1.83 g dibasic phosphate; 0.21 g monobasic phosphate potassium; 7.65 g sodium chloride; 1 L distilled water; pH 7.2) and mixed to obtain a ratio of Antibody producing cells: myeloma cells from 5:1 to 10:1, then centrifuged and the supernatant discarded. The cell pellet was thoroughly loosened and a mixed solution of polyethylene glycol 1000 (PEG-1000), MEM and dimethyl sulfoxide was added with stirring at 37°C. Then the MEM medium was added: first several times with an interval of 1-2 minutes, 1-2 ml, then to a total volume of 50 ml. Centrifuged, the supernatant was discarded, the cell pellet was carefully loosened and suspended in HAT medium (normal culture medium containing hypoxanthine, thymidine and aminopterin. The resulting suspension was cultured in an incubator with an atmosphere containing 5% CO ₂ at a temperature of 37 ° C for 7-14 days.

In addition, cell fusion can be carried out as follows. The spleen cells and myeloma cells are washed well with serum-free medium (eg, DMEM) or phosphate-buffered saline [hereinafter referred to as phosphate-buffered saline] and mixed at a spleen cell:myeloma cell ratio of about 5:1 to 10:1. then centrifuge. The supernatant is removed, the cell pellet is thoroughly loosened and 1 ml of serum-free medium containing 50% (wt/vol) polyethylene glycol with a molecular weight of 1000-4000 is added dropwise with stirring. Then 10 ml of serum-free medium is slowly added and then centrifuged. The supernatant is removed, the settled cells are suspended in a normal culture medium containing the required amount of hypoxanthine-aminopterin-thymidine (HAT) solution and human interleukin-2 (IL-2) (hereinafter referred to as HAT). The resulting suspension is added to the wells of the culture tablet (hereinafter referred to simply as the tablet) and cultured in an atmosphere containing 5% carbon dioxide at 37°C for 2 weeks. During the cultivation process, NAT medium is added as necessary.

(5) Hybridoma selection

If myeloma cells of a line with resistance to 8-azaguanine, that is, cells with hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, are used for fusion, then non-fused myeloma cells and myeloma cells that have fused with each other do not survive in the HAT medium. On the other hand, products of fusion of antibody-producing cells with each other, and hybridomas - products of fusion of antibody-producing cells with myeloma cells - can live in the NAT environment, but the lifespan of fused antibody-producing cells is significantly shorter. Thus, during long-term cultivation in the NAT medium, only hybridomas, the products of the fusion of antibody-producing cells with myeloma cells, survive, and thus selection of hybridomas is achieved.

In order for colonies to form from individual hybridomas, the culture medium is replaced: aminopterin is excluded from the NAT medium (hereinafter this medium is designated NT). A portion of the supernatant is collected from each resulting culture and the antibody-producing hybridoma can be selected using the method mentioned below for measuring antibody titer. In this way, hybridomas that produce antibodies are identified. Examples of methods for quantitative determination of antibody titer include various known methods such as radioimmunoassay (RIA method), enzyme-linked immunosorbent assay (ELISA method), fluorescent antibody labeling method and passive hemagglutination test; Of these, RIA or ELISA are preferable due to greater sensitivity, speed of execution, accuracy, automation capabilities, etc.

Those hybridomas that, based on the results of determining the antibody titer, produce the desired antibodies are transferred to another plate and cloned. Examples of cloning methods include the limiting dilution method, where the culture is diluted so that one well of the plate contains one cell, the soft agar method, where the culture is carried out in soft agar to collect colonies, the method in which individual cells are selected using a micromanipulator, the method , in which individual cells are selected using a cell sorter, , etc.

In the case of wells in which the antibody titer was observed, cloning is repeated 2-4 times, using, for example, the limiting dilution method; those cells that consistently show sufficient antibody titre are selected and used as a hybridoma strain producing monoclonal antibodies to TfR or a cell surface antigen, such as EGFR.

(6) Preparation of monoclonal antibodies

The monoclonal antibody-producing hybridomas obtained in (5) are administered by intraperitoneal injection to 8-10 week old mice or nude mice that have previously been injected with 0.5 ml of 2,6,10,14-tetramethylpentadecane (pristane) and then maintained for 2 weeks. After 10-21 days, the hybridoma causes the formation of an ascitic tumor. These tumors are removed from mice, the solid is separated by centrifugation, the remaining liquid is salted out with 40-50% ammonium sulfate and the precipitated material is purified by caprylic acid precipitation, on a DEAE-Sepharose or Protein A column or by gel filtration; IgG or IgM fractions are collected. In this way, purified monoclonal antibodies are obtained. It is also possible to grow a hybridoma in the abdominal cavity of a mouse, for example a linny BALB/c) or homozygous nude mouse (Nu/Nu), rat, hamster, guinea pig, rabbit or other animal, and monoclonal antibodies that bind to TfR or with a cell surface antigen such as EGFR.

After culturing the monoclonal antibody-producing hybridoma obtained in (5) in RPMI 1640 medium containing 10% FBS or another similar medium; the supernatant is separated by centrifugation, the pellet is suspended in GIT medium, SFM hybridoma medium containing 5% GF21 (from Daigo) or the like. The resulting suspension is cultivated for 3-7 days in a flask, by the roller method, in a culture bag or in another way. The resulting cell suspension is centrifuged, the supernatant is passed through a column with protein A or G, and the IgG fraction is collected. In this way, purified monoclonal antibodies are obtained. A simple purification method is also possible using commercially available monoclonal antibody purification kits, such as the MabTrap GII kit from Amersham Pharmacia Biotech, Inc. and so on.

The antibody subclass is determined by enzyme immunoassay using typing kits. Quantitative determination of protein is done using the Lowry method or calculated from absorbance at a wavelength of 280 nm [1.4( _OD280 ) = 1 mg immunoglobulin/ml].

(7) Monoclonal Antibody Binding Assay

with TfR or with a cell surface antigen such as (EGFR)

The binding activity of a monoclonal antibody to a TfR or to a cell surface antigen, such as EGFR, can be measured in a binding assay system such as Ouchterlony, ELISA, RIA, flow cytometry (FCM) or surface plasmon resonance (SPR).

The Ouchterlony method is easy to perform, but when the antibody concentration is low, concentration must be carried out. On the other hand, if you use the ELISA or RIA method, then provided that the supernatant obtained from the cell culture directly interacts with the antigen adsorbed on the solid phase and the subsequent use of secondary antibodies corresponding to various isotypes and subclasses of immunoglobulins, it is possible to identify the isotype and subclass of the desired antibody and quantify it binding activity.

As a specific example of an assay, a purified or partially purified recombinant cell surface antigen, such as EGFR or TfR, is adsorbed onto a solid phase in a 96-well ELISA plate, then that surface of the solid phase where there is no adsorbed antigen is blocked with an unrelated protein. a given antigen, such as bovine serum albumin (BSA). After ELISA, the plate is washed with phosphate buffered saline (PBS) and PBS containing 0.05% polysorbate 20 (Tween 20) (Tween-PBS) and reacted with serial dilutions of the first antibody (e.g., mouse serum, cell culture supernatant etc.), and these antibodies bind to the antigen immobilized on the solid phase. Then secondary antibodies are added to the wells of the plate - anti-immunoglobulin antibodies labeled with biotin, an enzyme (for example, horseradish peroxidase (HRP), alkaline phosphatase (ALP) or others), chemiluminescent agent, radioactive agent or otherwise, and these secondary antibodies bind to the first. After thoroughly washing the wells with Tween-PBS, a detection reaction is carried out according to the secondary antibody label taken and thus monoclonal antibodies that specifically interact with the target antigen are selected.

The FCM method allows you to measure the binding activity of a given antibody to cells in which the corresponding antigen is expressed [Cancer Immunol. Immunother. 36, 373 (1993)]. The binding of an antibody to an antigenic membrane protein on the cell membrane means that the antibody recognizes the conformation of the natural antigen and binds to it.

Examples of applications of the SPR method include kinetic analysis via Biacore. For example, using the Biacore T100, you can measure the kinetics of antigen binding to the test substance and analyze the data obtained using the software of this device. As a specific example of the procedure, After fixing the anti-mouse IgG antibody on the CM5 chip by immobilization at the amino group; introducing a stream of the test substance, such as hybridoma culture supernatant or purified monoclonal antibody, so that it binds in the appropriate amount; then a stream of antigen is injected at various known concentrations and binding and dissociation are measured. Kinetic analysis of the obtained data is carried out based on 1:1 binding using the instrument software and various kinetic parameters are determined. Alternatively, a cell surface antigen, such as EGFR or TfR, is captured onto the sensor chip, for example by amine immobilization, a stream of purified monoclonal antibody solution is injected at a variety of known concentrations, and binding and dissociation are measured. Kinetic analysis of the obtained data is carried out based on bivalent binding using the instrument software and various parameters are determined.

Also, the present invention can select antibodies that bind to TfR or a cell surface antigen, such as EGFR, in competition with an antibody against TfR or a cell surface antigen, such as EGFR, if the assay is performed in Biacore such that the analytes are present in the system described above for binding at the same time. Thus, by screening for antibodies whose binding to a given antigen is inhibited by the addition of a test antibody, it is possible to obtain antibodies that compete with the above antibody for binding to TfR or to a cell surface antigen, such as EGFR.

(8) Identification of monoclonal antibody epitope

to TfR or cell surface antigen, such as EGFR

In the present invention, the epitope that the antibody of the invention recognizes and binds is identified as follows.

For example, a partially defective version of the antigen is obtained. or a mutant variant of an antigen in which an amino acid residue that differs in different species is modified, or a mutant variant of an antigen in which a specific domain is modified, and if the antibody reactivity towards the defective or mutant variant is reduced, then this defective site or site where the amino acid has been modified represents the epitope to which the antibody is specific. Such a partially defective antigen variant or mutant antigen can be produced as a secretory protein by suitable host cells, for example E. coli, yeast, plant cells, or mammalian cells and the like; or such antigen can be produced in an antigen-expressing cell format by expressing it on the membrane of suitable host cells. In the case of a cell membrane-bound antigen, expression requires that the proper conformation of the antigenic protein be obtained, so expression on the host cell membrane is preferred. Also, to determine the reactivity of an antibody against a given antigen, a synthetic peptide can be obtained that imitates the primary structure or conformation of the antigen. For example, using known methods of peptide synthesis, various sections of the amino acid sequence of the antigen are obtained.

For example, in the case of the extracellular domain of a TfR or a cell surface antigen such as human or mouse EGFR, it is possible to identify the epitope to which the antibody is specific by creating a chimeric protein in which the domains representing the corresponding regions are specifically combined and testing for reactivity of a given antibody against that protein. It is also possible to determine in more detail the epitope to which a given antibody is specific by synthesizing various oligopeptides of the corresponding regions or producing mutant versions of the desired peptide using oligopeptide synthesis methods known to those skilled in the art, and then testing the reactivity of the antibody against these peptides. A simple way to prepare various oligopeptides is to use commercially available kits [eg, SPOTs Kit from Genosys Biotechnologies, Inc.; line of kits for multiple parallel synthesis of peptides (Multipin technology) manufactured by Chiron Corporation, etc.], which use Multipin technology or a similar method.

An antibody that binds to the same epitope as an antibody that binds to a TfR or a cell surface antigen, such as EGFR; can be obtained by identifying the epitope of the resulting antibody using the above-described binding assay system, then obtaining a synthetic peptide that is part of this epitope, a synthetic peptide that mimics the conformation of this epitope, or a recombinant epitope, or another variant thereof, and immunizing with the resulting peptide.

For example, if the epitope belongs to a membrane protein, then an antibody specific for that epitope can be more efficiently produced by creating a recombinant fusion protein in which all or part of the extracellular domain is attached to an appropriate tag, such as a FLAG tag or a histidine tag, or to the GST protein, or to the Fc region of an antibody, etc., and immunization is carried out with this recombinant protein.

2. Production of genetically engineered recombinant antibodies

Examples of obtaining genetically engineered recombinant antibodies are generally described in the works of P. J. Delves., Antibody Production Essential Techniques., 1997 Wiley; P. Shepherd, C. Dean. Monoclonal Antibodies., 2000 Oxford University Press; J. W. Goding., Monoclonal Antibodies: principles and practice., 1993 Academic Press, and elsewhere; however, methods for producing chimeric antibodies, humanized antibodies and human antibodies are described below herein. Also, using the same methods, genetically engineered recombinant antibodies can be obtained from mice, rats, hamsters and rabbits.

(1) Preparation of cDNA encoding the V region of a monoclonal antibody produced by a hybridoma

Complementary DNA (cDNA) encoding the VH and VL of a monoclonal antibody is prepared, for example, as follows.

First, mRNA is isolated from a hybridoma producing a monoclonal antibody and cDNA is synthesized from it. The synthesized cDNAs are then cloned individually into a vector, such as a phage or plasmid, thereby obtaining a cDNA library. From this library, a recombinant phage or a recombinant plasmid containing cDNA encoding VH or VL is isolated using DNA encoding the antibody constant or variable region, respectively, as a probe. The complete nucleotide sequence encoding VH or VL in the isolated recombinant phage or recombinant plasmid is determined, and then the complete amino acid sequence of VH or VL is deduced from the nucleotide sequence.

Non-human animals, such as mice, rats, hamsters, rabbits, or the like, are usually used to produce hybridomas, but any animal can be used as long as the resulting hybridoma can be obtained.

To obtain total RNA from a hybridoma, a method using guanidine thiocyanate and cesium trifluoroacetate is used [Methods in Enzymol., 154, 3 (1987)] or a ready-made kit, for example RNA easy Kit (manufactured by QIAGEN, Inc.) or the like.

Messenger RNA is isolated from total RNA using oligo(dT)-cellulose columns [Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989)] or using ready-made kits such as Oligo-dT30 <Super> mRNA Purification Kit (manufactured by Takara Bio, Inc.), etc. To obtain mRNA, you can also use kits such as the Fast Track mRNA Isolation Kit (manufactured by Invitrogen, Inc.) or the QuickPrep mRNA Purification Kit (manufactured by Pharmacia Corporation).

To synthesize cDNA and prepare a cDNA library, a known method is used, described, for example, in Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997), or use ready-made kits, for example SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning, (manufactured by Invitrogen, Inc.) or ZAP-cDNA Synthesis Kit (manufactured by Stratagene Corporation), etc.

Once a cDNA library has been obtained, any vector into which this cDNA can be inserted can be used as a vector into which the cDNA synthesized from the mRNA isolated from the hybridoma is inserted.

For example, suitable vectors include ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], λZAP II (manufactured by Stratagene Corporation), λgt 10, λgt 11 [DNA Cloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid (manufactured by Clontech Laboratories, Inc.), λExCell, pT7T3-18U (manufactured by Pharmacia Corporation), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)] or the like.

The E. Coli into which the cDNA library created with a phage or plasmid vector is introduced can be any E. coli capable of incorporating, expressing and maintaining the cDNA library. For example, strains such as XL1-Blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], can be used. NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)], JM105 [Gene, 38, 275 (1985)] or the like.

In order to select cDNA clones encoding non-human VH or VL antibodies from a cDNA library, colony hybridization using radioactively or fluorescently labeled probes or plaque hybridization is used [Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989)] or similar methods.

In addition, cDNA encoding VH or VL can also be obtained by polymerase chain reaction (hereinafter referred to as PCR) by selecting appropriate primers [Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)] and using cDNA synthesized from mRNA or a cDNA library as a template.

Selected cDNAs are digested with suitable restriction enzymes or bypass enzymes, then cloned into a plasmid, for example pBluescript SK(-) (manufactured by Stratagene Corporation), and the nucleotide sequence of the cDNA is determined by commonly used nucleotide sequence analysis methods. For example, terminating dideoxynucleotides are used [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or carry out automatic determination of the nucleotide sequence, for example using an A.L.F. sequencer. DNA sequencer (manufactured by Pharmacia Corporation).

Based on the established complete nucleotide sequence, the complete amino acid sequence of VH or VL is derived and compared with the complete amino acid sequence of the corresponding VH and VL of a known antibody [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]; in this way, it is determined whether the resulting cDNA encodes the full-length amino acid sequence of the VH or VL antibody containing the signal peptide required for secretion.

Having the complete amino acid sequences of the VH and VL of the antibody containing the secretion signal sequence, the complete amino acid sequence is compared with the VH and VL of a known antibody [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] and thus determine the length of the secretion signal sequence, , and N-terminal amino acid sequence, as well as which subgroup they belong to.

In addition, by comparing the VH and VL sequences with the sequences of a known antibody, the amino acid sequences of the CDRs in VH and VL can be determined [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].

Also, having the obtained complete amino acid sequences of VH and VL, it is possible to determine whether the specified VH and VL sequences are new, for example, by conducting a homology search, for example, using the BLAST computer programs [J. Mol. Biol., 215, 403 (1990)] or similar, in databases such as SWISS-PROT or PIR-Protein..

(2) Construction of an expression vector to obtain

genetically engineered recombinant antibody

An expression vector for genetic engineering can be obtained by cloning DNA encoding at least CH or CL of a human antibody into an expression vector suitable for use in an animal cell.

As the C regions, the CH and CL of an arbitrary human antibody, for example, the CH of the γ1 subclass and the CL of the κ subclass of a human antibody or the like, can be used. cDNA is used as DNA encoding the CH or CL of a human antibody, but chromosomal DNA, which contains exons and introns, is also suitable.

Any vector can be used as an expression vector for animal cells, provided that it can include the gene encoding the C region of a human antibody and express it; for example, suitable vectors are pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)], pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)], INPEP4 (manufactured by Biogen-IDEC, Inc.) , N5KG1val (US Patent No. 6,001,358), N5KG4PE R409K (described in WO 2006/033386), N5KG2 vectors (described in WO 2003/033538), transposon vectors (WO 2010/143698), etc.

The SV40 early promoter can serve as a promoter and enhancer in an expression vector for animal cells [J. Biochem., 101, 1307 (1987)], (LTR) Moloney murine leukemia virus [Biochem. Biophys. Res. Commun., 149, 960 (1987)], CMV promoter (US Pat. No. 5,168.06) or promoter [Cell, 41, 479 (1985]) and enhancer [Cell, 33, 717 (1983)] of the immunoglobulin H chain gene or similar regulatory elements.

For the expression of a genetically engineered recombinant antibody, in order to facilitate the construction of the vector, its introduction into animal cells, the equilibrium ratio between the levels of expression of the H- and L-chains of the antibody, and for other reasons, a vector containing genes of both the L- and H-chain ( tandem type vector) [J. Immunol. Methods, 167, 271 (1994)], but you can also use a combination of several vectors, each of which carries one gene - H- or L-chains (vectors)

To obtain genetically engineered recombinant antibodies, tandem-type expression vectors such as pKANTEX93 (WO 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], N5KG1val (USA patent No. 6,001,358), N5KG4PE R409K (described in WO 2006/033386), N5KG2 vectors (described in WO 2003/033538), Tol2 transposon vector (WO 2010/143698) or the like.

(3) Construction of an expression vector to obtain

chimeric antibody

To make an expression vector to produce a chimeric antibody, the cDNA encoding a non-human VH or VL antibody generated in (1) is cloned into the expression vector to produce a recombinant antibody constructed in (2) by placing it in front of each of the genes encoding CH or human antibody CL.

First, to ligate the cDNA encoding the VH or VL of a non-human antibody at the 3' end with the CH or CL sequences of a human antibody located at the 5' end, cDNAs encoding the VH and VL are prepared so that the nucleotide the sequence of the ligation site encodes the corresponding amino acids and there is a sequence recognized by a suitable restriction enzyme. Then, each of the resulting VH and VL cDNAs is cloned into an expression vector to produce the recombinant antibody constructed in (2) upstream of the gene encoding the human antibody CH or CL, respectively, so that they are expressed in the desired form; thus obtaining an expression vector for the chimeric antibody.

In addition, each of the cDNAs encoding non-human VH or VL antibodies is amplified by PCR using synthetic DNA containing at both ends the corresponding nucleotide sequence recognized by a restriction enzyme, and cloned into an expression vector to obtain a genetically engineered recombinant antibody designed at 2); in this way an expression vector for the chimeric antibody is also obtained.

(4) Preparation of cDNA encoding the V region

humanized antibody

cDNA encoding the VH or VL of the humanized antibody is prepared as follows. First, those amino acid sequences of framework regions (hereinafter referred to as (FR)) of the VH or VL of the human antibody are selected to which the amino acid sequences of the CDR VH or VL of the non-human antibody obtained in (1) are to be grafted.

As the FR amino acid sequence, any amino acid sequence derived from a human antibody can be selected. For example, the amino acid sequence of a human antibody FR recorded in a database such as the Protein Data Bank, or an amino acid sequence typical of a given subset of human antibody FRs [Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991)] may be used. and so on. To avoid reduction in antibody-antigen binding activity, human antibody FR amino acid sequences are selected that are as homologous as possible (with a degree of homology of 60% or more) to the amino acid sequences of the corresponding FRs in the VH or VL of the parent non-human antibody.

Each of the CDR amino acid sequences of the original non-human antibody is then grafted onto a selected FR amino acid sequence in the VH or VL of the human antibody and the VH or VL amino acid sequences of the humanized antibody are constructed. From the compiled amino acid sequence, a nucleotide sequence is derived, taking into account codon preference, characteristic of the nucleotide sequence of the gene of a given antibody [Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991)], and the cDNA sequence for the VH or VL of the humanized antibody is compiled.

Based on the compiled cDNA sequence, several DNAs 100-150 nucleotides long are synthesized and PCR is performed using them. In this case, for the sake of the efficiency of the PCR reaction and taking into account the length of the synthesized DNA, 4-6 synthetic DNAs are obtained for each heavy and each light chain. It is also possible to synthesize and use synthetic DNA encoding the full-length variable region.

Next, a nucleotide sequence recognized by a suitable restriction enzyme is inserted at the 5’ end of the synthetic DNA on both sides, which allows cloning of the cDNA encoding the VH or VL of the humanized antibody in an expression vector to obtain a genetically engineered recombinant antibody according to y (2). After PCR, each amplified product is cloned into a plasmid vector, for example pBluescript SK(-) (manufactured by Stratagene Corporation), the nucleotide sequence is determined as described in (1), and thus a plasmid is obtained containing DNA encoding the amino acid sequence of the VH or VL of the desired humanized antibody.

(5) Amino acid sequence modification

V region of a humanized antibody

The antigen binding activity of a humanized antibody produced by grafting only the VH and VL CDRs of a non-human antibody onto the VH and VL framework regions of a human antibody is lower than that of the parent non-human antibody. [BIO/TECHNOLOGY, 9, 266 (1991)]. In this regard, the reduced antigen binding activity of such a humanized antibody can be enhanced; To do this, it is necessary to establish which amino acid residues in the FR sequences of the VH or VL regions of a human antibody are directly involved in antigen binding, which interact with the CDR and which ensure the proper conformation of the antibody molecule and are involved in antigen binding indirectly, and based on this, replace these amino acid residues to those that correspond to them in the polypeptide chains of antibodies of non-human origin.

To identify the FR amino acid residues involved in antigen binding, the spatial structure of the antibody molecule is reconstructed and analyzed by X-ray crystallography [J. Mol. Biol., 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)] or other methods. It is also possible to obtain a modified humanized antibody with the desired antigen-binding activity through trial and error: making several different modifications for each antibody and testing how these modifications relate to antigen-binding activity.

The amino acid residues of the FR regions of the VH or VL of a human antibody can be replaced by performing the PCR described in (4); in this case, synthetic DNA is used for changes. After PCR, the nucleotide sequence of the amplification product is determined as described in (1) to ensure that the desired changes have occurred.

(6) Expression vector construction

to obtain a humanized antibody

An expression vector for producing a humanized antibody is constructed by cloning each of the cDNAs encoding the VH or VL of a composed humanized antibody before the corresponding genes encoding CH or CL of a human antibody in the expression vector for producing a genetically engineered recombinant antibody as in (2).

For example, in an expression vector to obtain a genetically engineered recombinant antibody according to point (2), before each of the genes encoding CH or CL of a human antibody, a nucleotide sequence recognized by a suitable restriction enzyme is inserted at the 5' end of the synthetic DNA on both sides from the number synthetic DNA used in the construction of the VH or VL humanized antibody obtained under paragraph (4) or (5); as a result, they are expressed in the proper form.

(7) Expression vector construction

to obtain human antibodies

When a monoclonal antibody-producing hybridoma is prepared using one that produces human antibodies as an immunized animal, it is possible to determine the amino acid sequences and cDNA sequences for the VH and VL of the human antibody according to (1). And then it is possible to construct an expression vector for producing a human antibody by cloning each of the genes encoding VH or VL of the human antibody obtained in (1) before each of the genes encoding CH or CL of the human antibody in the expression vector to obtain a genetically engineered recombinant antibody , obtained from (2).

(8) Transient expression of genetically engineered recombinant antibody

The antigen binding activity of many different engineered recombinant antibodies can be advantageously determined by transiently expressing the engineered recombinant antibody using an expression vector to produce the engineered recombinant antibody obtained in (3), (6) and (7) or expression the vector obtained as a result of modification of the specified vector.

Any cell capable of expressing a genetically engineered recombinant antibody, such as COS-7 cells (ATCC accession number CRL1651), is suitable as a host cell for introducing the expression vector. To introduce the expression vector into COS-7 cells, transfection using DEAE-dextran is used [Methods in Nucleic Acids Res., CRC press (1991)], lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and other similar methods.

After introduction of the expression vector into the host cells, the expression level of the genetically engineered recombinant antibody and its antigen binding activity are determined in the culture supernatant using an enzyme-linked immunosorbent assay [Monoclonal Antibodies-Principles and practice, 3rd edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experimental Manual, Kodansha scientific books (1987)] or other method.

(9) Obtaining a cell line with stable expression

genetically engineered recombinant antibody and production of genetically engineered recombinant antibody

Transformed cells with stable expression of the engineered recombinant antibody are obtained by introducing the expression vector for producing the engineered recombinant antibody of (3), (6) and (7) into suitable host cells.

To introduce an expression vector into host cells, for example, electroporation [JP-A-H2-257891, Cytotechnology, 3, 133 (1990)], treatment with calcium ions, and spheroplasts, lithium acetate, calcium phosphate, lipofection, and others are used. similar methods. In addition, the desired gene can be introduced into the animal’s body (see below), for example, by microinjection; a method of introducing a gene into ES cells by electroporation or lipofection, as well as nuclear transfer and other methods.

The host cells into which the expression vector is introduced to produce the engineered recombinant antibody can be any cell capable of expressing the engineered recombinant antibody. For example, suitable mouse cells ES SP2/0-Ag14 (ATCC CRL 1581), P3X63-Ag8.653 (ATCC CRL 1580), Chinese hamster cells CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro -5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat No. 11619), Dihydrofolate reductase-deficient CHO (CHO/DG44) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO-Lec13 with acquired lectin resistance [Somatic Cell and Molecular Genetics, 12, 55 (1986)], CHO with α1,6-fucosyltransferase deficiency (WO 2005/035586, WO 02/ 31140), rat cells YB2/3HL.P2.G11.16Ag.20 (ATCC CRL 1662) and other similar cells.

It is also possible to use host cells in which the activity of a specific protein is reduced or lost, for example an enzyme involved in the intracellular synthesis of the sugar derivative of the nucleotide GDP-fucose; or an enzyme involved in the modification of the sugar chain, for example providing an α1-6 bond between fucose and N-acetylglucosamine molecules at the reducing end of the N-glycosidic carbohydrate chain; or a protein involved in the transport of GDP-fucose in the Golgi apparatus, or other similar proteins; for example, α1,6-fucosyltransferase-deficient CHO cells are suitable (WO 2005/035586, WO 02/31140).

After introduction of the expression vector into host cells, transformants with stable expression of the engineered recombinant antibody are selected by culturing the resulting transformed cells in animal cell culture medium containing the drug, for example G418 sulfate (JP-A-H2-257891).

For culturing animal cells according to this invention, media such as RPMI 1640 (manufactured by Invitrogen, Inc.), GIT (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL 301 (manufactured by JRH Biosciences, Inc.), EX-CELL are used 302 (manufactured by JRH Bioscience, Inc.), EX-CELL 325 (manufactured by JRH Bioscience., Inc.), IMDM (manufactured by Invitrogen, Inc.) or Hybridoma-SFM (manufactured by Invitrogen, Inc.), or these media supplemented with various ingredients, such as FBS, or other similar media. Selected transformants are cultured in a suitable medium, and a genetically engineered recombinant antibody is expressed in the cells, which accumulates in the culture medium. In the supernatant of the culture medium, the level of expression of the genetically engineered recombinant antibody and its antigen binding activity are quantified using ELISA or other methods. In addition, the expression of the engineered recombinant antibody in the transformed cells can be enhanced by using the DHFR amplification system (JP-A-H2-257891) or the like.

To purify a genetically engineered recombinant antibody from the supernatant of a transformed cell culture, columns with protein A are used [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. Protein purification methods such as gel filtration, ion exchange chromatography and ultrafiltration can also be combined for purification.

To determine the molecular weight of the H- and L-chains and the whole molecule of a purified genetically engineered recombinant antibody, polyacrylamide gel electrophoresis is used [Nature, 227, 680 (1970)], or Western blotting [Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], or other methods.

3. Design of bispecific antibodies

The bispecific antibody of the present invention can be created by constructing each of the N-terminal side polypeptides and the IgG portion, and constructing a bispecific antibody where they are combined.

3-1. Construction of the IgG part

To make the IgG part, monoclonal antibodies are prepared in the manner described in 1 above; For each antibody, cDNA sequences encoding the CDR and variable region are determined as described in 2 above, and an IgG portion including the CDR or variable region of the antibody is constructed.

3-2. Construction of the N-terminal side polypeptide

When the CDR or variable region of an antibody is included in an N-terminal side polypeptide, to obtain it, the nucleotide sequences encoding the CDR or variable region of the antibody are determined according to the method as described in 1 and 2 above, and an N-terminal side polypeptide incorporating them is constructed. The N-terminal side polypeptide may be single chain, in which VH and VL are connected directly or through an appropriate linker into a scFV or the like; or expressed in double-stranded form, for example, Fab, dsFv and the like structures, where the S-S bond is formed after expression; or have a structure like VHH or similar. In the resulting polypeptide variants of the N-terminal side, the antigen binding activity is determined by the methods described above and the one that possesses it to the proper extent is selected.

4. Obtaining bispecific antibodies

4-1. Preparation of bispecific antibodies with general light chains

A bispecific antibody in which the N-terminal polypeptide is a Fab and the light chain has a structure common to the Fab and the IgG portion is prepared as follows.

DNA is synthesized encoding a polypeptide in which the VH-CH1 of the polypeptide of the N-terminal side is connected to the VH part of the IgG, as well as DNA encoding the VL of each antibody; these DNAs are inserted into the expression vector for producing the genetically engineered recombinant antibody described in 2. (2) above, and expressed, thereby obtaining a bispecific antibody. In the case where the IgG portion and the polypeptide of the N-terminal side are connected via a linker, DNA encoding the linker is also synthesized.

4-2. Preparation of a bispecific antibody in which the N-terminal polypeptide includes non-general light chains containing CDRs

(1) A non-general type light chain bispecific antibody in which the N-terminal side polypeptide is Fab is prepared as follows.

DNA encoding a polypeptide in which the VL-CL polypeptide of the N-terminal side is connected to the VH part of the IgG, as well as DNA encoding the VH-CH1 polypeptide of the N-terminal side and DNA encoding the VL part of the IgG are synthesized; these DNAs are included in the expression vector for producing a genetically engineered recombinant antibody described in 2. (2) above and expressed, thereby obtaining a bispecific antibody. In the case where the IgG portion and the polypeptide of the N-terminal side are connected via a linker, DNA encoding the linker is also synthesized.

(2) A bispecific antibody in which the N-terminal side polypeptide is VHH is prepared as follows.

DNA encoding a polypeptide in which the VHH and VH parts of IgG are combined, as well as DNA encoding the VL parts of IgG are synthesized; these DNAs are included in the expression vector for producing a genetically engineered recombinant antibody described in 2. (2) above and expressed, thereby obtaining a bispecific antibody. In the case where VHH and the IgG part are connected via a linker, DNA encoding the linker is also synthesized.

(3) A bispecific antibody in which the N-terminal side polypeptide is a polypeptide including scFv, dsFv or CDR and different from those described in (1) and (2) above is prepared as follows.

If the N-terminal side polypeptide is single-stranded, then DNA is synthesized in which DNA encoding the N-terminal side polypeptide is combined with DNA encoding the VH portion of the IgG. If the N-terminal side polypeptide is a complex consisting of two single-stranded polypeptides, then synthesize DNA encoding one of the single-stranded polypeptides forming the N-terminal side polypeptide, which is connected to DNA encoding the VH part of the Ig, as well as DNA encoding the other single-stranded a polypeptide forming an N-terminal polypeptide. In addition, DNA encoding the VL portion of IgG is synthesized. In the case where the IgG portion and the polypeptide of the N-terminal side are connected via a linker, DNA encoding the linker is also synthesized. These DNAs are included in the expression vector for producing a genetically engineered recombinant antibody described in 2. (2) above and expressed, thereby obtaining a bispecific antibody.

4-3. Preparation of a bispecific antibody comprising an N-terminal polypeptide other than those described above

When the N-terminal polypeptide is different from those described above, the bispecific antibody of the present invention is prepared as follows.

If the N-terminal polypeptide is single-stranded, then DNA is synthesized in which DNA encoding the polypeptide of the N-terminal side and DNA encoding the VH portion of the IgG are combined. If the N-terminal polypeptide is represented by a complex consisting of two or more single-chain polypeptides, then the synthesized DNA encoding one of the single-chain polypeptides forming the polypeptide of the N-terminal side is combined with DNA encoding the VH parts of IgG, and DNA encoding the remaining single-stranded ones is also synthesized polypeptides that form the N-terminal side of the polypeptide. In addition, DNA encoding the VL portion of IgG is synthesized. In the case where the IgG portion and the polypeptide of the N-terminal side are connected via a linker, DNA encoding the linker is also synthesized. These DNAs are included in the expression vector for producing a genetically engineered recombinant antibody described in section 2.(2) above and expressed, thereby obtaining a bispecific antibody.

These DNAs are included in the expression vector for producing a genetically engineered recombinant antibody described in 2. (2) above and expressed, thereby obtaining a bispecific antibody. In the case where the IgG portion and the polypeptide of the N-terminal side are connected via a linker, DNA encoding the linker is also synthesized.

The antigen binding domain can be prepared and isolated by methods such as phage or yeast display other than the hybridoma method described above in 1 [Emmanuelle Laffy et. al., Human Antibodies 14, 33-55, (2005)].

In cases where a bispecific antibody consisting of several VHs and one VL (hereinafter referred to as a general light chain bispecific antibody) is to be produced, screening is carried out by phage display or a similar method and the VH chains most suitable for the target are selected. given VL, so that in the resulting bispecific antibody, each antigen-binding domain will interact with a specific antigen.

Namely, first, an animal is immunized with the first antigen as described in 1 above, and hybridomas are obtained from the spleen of the immunized animal; the nucleotide sequence encoding the first antigen binding domain is then cloned. The animal is then immunized with a second antigen and a cDNA library is obtained from its spleen; DNA encoding the VH amino acid sequence is then obtained from this library using PCR.

After this, a phage library is obtained expressing scFvs in which the VH obtained as a result of immunization with the second antigen and the VL of the first antigen-binding domain are connected; by panning a phage library, phages exhibiting scFvs that specifically bind to the second antigen are selected. Using the selected phage, DNA encoding the amino acid sequence of the VH second antigen binding domain is cloned.

If the N-terminal side polypeptide is Fab, then DNA encoding the amino acid sequence of the polypeptide is constructed, in which the VH of the first antigen binding domain and the VH of the second antigen binding domain are connected through a CH1 or CH1 region and a linker; this DNA and the DNA encoding the amino acid sequence of a single VL are inserted, for example, into an expression vector to produce the genetically engineered recombinant antibody described in 2 above. (2) thus obtaining an expression vector for the bispecific antibody of the present invention.

5. Determination of the activity of bispecific antibodies or fragments of this antibody according to the invention

Determination of the activity of purified bispecific antibodies or bispecific fragments of a given antibody is carried out as follows.

The binding activity of the bispecific antibodies of the present invention to a cell line expressing at least a TfR or a cell surface antigen such as EGFR, or both, can be measured using the binding assay system described in 1. (7) above.

CDC activity or ADCC activity directed against cells expressing at least TfRα or a cell surface antigen, for example EGFR, or both, is determined by measurement methods known in the art [Cancer Immunol. Immunother. 36, 373 (1993)].

The "anti-cellular" activity (also called growth inhibitory activity) of the bispecific antibodies of the present invention can be measured in the following manner. For example, cells are seeded in a 96-well plate, antibodies are added and the cells are cultured for a while, after which the WST-8 reagent (manufactured by Dojindo Molecular Technologies, Inc.) is added, the reaction is allowed to occur, and the absorbance is measured at a wavelength of 450 nm using a plate reader. ; The data obtained allows us to assess cell viability.

Intracellular signaling of a TfR or cell surface antigen, such as EGFR, is determined by detecting phosphorylation of proteins in the cell by Western blotting or the like.

6. Method for treating diseases using bispecific antibodies or fragments of this antibody according to the invention

The bispecific antibodies or bispecific antibody fragments of this invention can be used to treat diseases associated with at least TfR or a cell surface antigen, such as EGFR, or both; This preferably concerns diseases in which cells that express TfR and a cell surface antigen, such as EGFR, play a role. An example of diseases associated with at least TfR or a cell surface antigen such as EGFR, or both, are malignancies, cancers, and the like.

Examples of malignant tumors and cancers include colon cancer, colorectal cancer, lung cancer, breast cancer, glioma, malignant melanoma, thyroid cancer, renal cell carcinoma, leukemias, lymphomas, T-cell lymphoma, gastric cancer, pancreatic cancer, cervical cancer uterus, 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, laryngeal cancer , pleural tumors, arrhenoblastoma, endometrial hyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi's sarcoma, angioma, cavernous hemangioma, angioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, glioblastoma, osteogenic sarcoma coma, leiomyosarcoma, Wilms tumor and so on.

The therapeutic agent containing the bispecific antibody or bispecific antibody fragment of the present invention, or derivatives thereof, may contain only said antibody, or antibody fragment, or derivatives thereof as the active ingredient, however, the therapeutic agent represented by the pharmaceutical preparation obtained is generally preferred. methods known in the pharmaceutical field involving mixing the active ingredient with one or more pharmacologically acceptable carriers.

Examples of routes of administration of the therapeutic agent of this invention include oral or parenteral administration, such as oral, airway, rectal, subcutaneous, intramuscular, and intravenous. Examples of dosage forms of the therapeutic agent of the present invention include aerosols and sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injectables, ointments, patches, and the like. Various pharmaceutical preparations of the therapeutic agent of this invention can be prepared by methods conventional for such purposes, including the use of excipients, fillers, binding agents, wetting agents, disintegrating agents, surfactants, glidants, dispersing agents, buffering agents, preservatives, solubilizing agents, antiseptics, dyes, flavors, stabilizing agents, etc.

Examples of excipients include lactose, fructose, glucose, corn starch, sorbitol, crystalline cellulose, sterile water, ethyl alcohol, glycerin, physiological saline, buffer solutions and the like. Examples of disintegrants include starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, synthetic magnesium silicate and the like.

Examples of binders include methylcellulose or its salts, ethylcellulose, gum acacia, gelatin, hydroxypropylcellulose, polyvinylpyrrolidone, etc. Examples of glidants include talc, magnesium stearate, polyethylene glycol, hydrogenated vegetable oil, etc.

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

Examples of other additional ingredients include syrup, petrolatum, glycerin, ethyl alcohol, propylene glycol, citric acid, sodium chloride, sodium nitrite, sodium phosphate, etc.

Examples of pharmaceutical preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules, etc.

Liquid pharmaceuticals, such as emulsions or syrups, use water to make them; sugars, such as sucrose, sorbitol or fructose; glycols, such as polyethylene glycol or propylene glycol; oils such as sesame, olive or soybean oil; preservatives, for example para-hydroxybenzoic acid ester; flavorings such as strawberry or mint; and other additives.

For the manufacture of capsules, tablets, powders, granules and similar preparations, excipients such as lactose, glucose, sucrose or mannitol are used; disintegrants such as starch or sodium alginate; glidants such as magnesium stearate or talc; binding agents such as polyvinyl alcohol, hydroxypropylcellulose or gelatin; surfactants, for example fatty acid esters; plasticizing agents such as glycerin; and other similar additives.

Examples of pharmaceutical preparations suitable for parenteral administration include injectable preparations, suppositories, sprays, etc. For the manufacture of injectable preparations, the carriers used are saline solution, glucose solution or a mixture of these two solutions.

To make suppositories, carriers such as cocoa butter, hydrogenated fats or carboxylic acids are used. For the manufacture of sprays, carriers are used that do not irritate the mucous membrane of the oral cavity or airways of the patient and dispersing monoclonal antibodies or antibody fragments according to this invention in the form of small particles. which promotes their absorption, etc. Examples of such carriers include lactose, glycerin, etc. Also, the therapeutic agent of this invention can be presented in the form of an aerosol or dry powder. In addition, some of the additive components used in oral pharmaceutical preparations may also be used in the above-mentioned parenteral preparations.

The effective dosage of the drug according to this invention, consisting of an effective amount of bispecific antibodies and the required amount of diluent and pharmacologically acceptable carrier, ranges from 0.0001 mg per 1 kg of body weight to 100 mg/kg per dose, and the time interval between administrations ranges from 2 days to 8 weeks.

7. Method for diagnosing diseases using bispecific antibodies or bispecific antibody fragments according to this invention

Detection or quantification of cells that express at least TfR, or a cell surface antigen, such as EGFR, or both, using bispecific antibodies or bispecific antibody fragments of the present invention allows the diagnosis of diseases associated with at least TfR, or with a cell surface antigen, eg EGFR, or both, preferably diseases in which cells expressing TfR and a cell surface antigen, eg EGFR, play a role.

Diagnosis of diseases such as malignancies and cancers that are associated with at least TfR or a cell surface antigen, such as EGFR, or both, can be accomplished, for example, by detecting or quantifying TfR and/or cell surface antigen , for example EGFR, as follows.

First, biological samples from a variety of healthy individuals are examined; to detect or quantify their TfR and/or cell surface antigen, for example EGFR, using the immunoassay methods described below using bispecific antibodies, or bispecific antibody fragments of the present invention, or derivatives thereof; analyze the obtained data on the amount of TfR and/or cell surface antigen, for example EGFR, in biological samples from healthy individuals.

The same methods are then used to determine the amount of TfR and/or cell surface antigen, such as EGFR, in the biological sample of the test subject and compare the obtained data with those of healthy individuals. If the test subject has an increased amount of TfR and/or cell surface antigen, such as EGFR, compared to healthy individuals, then he is diagnosed with cancer. Other diseases associated with TfR and/or cell surface antigen, such as EGFR, are diagnosed in the same way.

Immunological methods are methods for detecting or quantifying an antibody or antigen using labeled antigen or antibody, respectively. Examples of such methods include assays that use radiolabeled antibodies; linked immunosorbent assay; analysis using fluorescent or luminescent labels, Western blotting, physicochemical methods, etc.

Examples of assays using radiolabeled antibodies include a method in which a bispecific antibody or bispecific antibody fragment of the present invention reacts with an antigen, or a cell where the antigen is expressed, or the like, then reacts with an anti-immunoglobulin antibody or a binding fragment thereof carrying radioactive tracer, and is detected using a scintillation counter or similar measuring device.

Examples of the enzyme immunoassay include a method in which a bispecific antibody or a bispecific antibody fragment of the present invention reacts with an antigen, or a cell where the antigen is expressed, or the like, then reacts with an anti-immunoglobulin antibody or a binding fragment thereof carrying a label, and with subsequent measurement of color using an absorptiometer. An example of such a method is the sandwich ELISA.

Known enzyme labels are used as labels for enzyme immunoassay [IGAKU-SHOIN Ltd. (1987)], for example, alkaline phosphatase, peroxidase, luciferase; Biotin tag, etc. is also used.

In the sandwich version (ELISA, an antibody is immobilized on the solid phase, to which the antigen that is the target of the analysis binds, and then a second antibody interacts with the antigen that has bound the first antibody. ELISA uses two types of antibodies, which are an antibody or a fragment specific to the antigen that needs to be detected or quantified, and having different antigen-binding domains: the first is antibodies or antibody fragments that are pre-adsorbed on a plate (for example, 96-well); the second antibodies or antibody fragments are pre-labeled with a fluorescent agent, e.g. FITC, an enzyme, for example peroxidase, biotin, etc. Cells or tissues or their homogenates taken from the body; cell culture supernatant; blood serum; pleural effusion; ascites fluid; intraocular fluid, etc. are applied to a plate with immobilized first antibodies, then Labeled antibodies or antibody fragments are added and a detection reaction occurs in accordance with the label. For quantitative determination, a calibration curve is constructed from analysis data with serial dilutions of an antigen of known concentration, which is used to calculate the antigen content in the sample.

The sandwich ELISA can use polyclonal antibodies, monoclonal antibodies, and antibody fragments such as Fab, Fab', or F(ab) ₂ . The two types of antibodies in a sandwich ELISA can be a combination of monoclonal antibodies or antibody fragments that bind to different epitopes, or a combination of polyclonal and monoclonal antibodies or antibody fragments.

An example of a fluorescent and immunological assay is the method described in: Monoclonal Antibodies - Principles and Practice, 3rd edition, Academic Press (1996); Monoclonal Antibody Experimental Manual, Kodansha scientific books (1987), etc. Well-known fluorescent agents are used as labels [Fluorescent Antibody Method, Soft Science, Inc. (1983)], such as FITC, RITC, etc.

An example of a luminescent immunoassay is the method described in Bioluminescence and Chemiluminescence Clinical Test 42, Hirokawa-Shoten Ltd. (1998) and other similar sources. In this analysis, well-known luminescent agents are used as labels, for example acridine ester, triphenylimidazole (lofin), etc.

The Western blot method consists of the following: the antigen, or a cell with an antigen, or other substance is fractionated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) [Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory (1988)]. Blots are then used to transfer the molecules from the gel onto a polyvinylidene fluoride (PVDF) or nitrocellulose membrane, allow the membrane to react with an antibody or antibody fragment that binds to the antigen of interest, and then allow it to react with an anti-IgG antibody or antibody fragment labeled with a fluorescent agent, e.g. FITC, or an enzyme such as peroxidase, or biotin, or other detectable agent, followed by visualization of the label. An example of such an analysis is described below.

Cells or tissue expressing a polypeptide with the desired amino acid sequence are lysed and the lysate is analyzed by SDS-PAGE under reducing conditions, loading the analyte at 0.1-30 μg of protein per lane. After electrophoresis, the protein is transferred to a PVDF membrane and the sites are blocked with a PBS solution containing 1-10% BSA (hereafter referred to as BSA-PBS) for 30 minutes at room temperature. Then react with the bispecific antibodies of this invention, after which the membrane is washed with PBS containing 0.05 - 0.1% Tween 20 (Tween-PBS)), and treated with peroxidase-labeled goat anti-mouse IgG antibodies for 2 hours at room temperature. Wash the membrane with Tween-PBS and detect the band to which the antibodies are bound using ECL Western Blotting Detection Reagents (manufactured by Amersham, Inc.) or similar detection agent; in this way the desired antigen is detected. Western blotting uses antibodies that can bind to a polypeptide that has not retained its natural conformation.

Physicochemical methods include, for example, those that detect an aggregate formed by the binding of at least one antigen, namely TfR or a cell surface antigen, for example EGFR, with a bispecific antibody or bispecific antibody fragment. Another example of a physicochemical method is the capillary tube method, one-dimensional immunodiffusion, immunoturbidimetric analysis, including latex microparticles [Outline of Clinical Examination Method, KANEHARA & Co., LTD. (1998)] and other similar methods.

In latex immunoturbidimetry, carrier particles, for example polystyrene latex, with a diameter of about 0.1-1 μm are sensitized by attaching antibody (or antigen) molecules to it, so that the formation of an antibody-antigen complex occurs on the particles, due to which the light in the reaction mixture is scattered more strongly, and less passes through it. The concentration of the antigen in the sample is determined by the change in absorption or scattering of light caused by interaction with the antibody specific to it on the particles.

Known immunological methods can be used to identify or quantify cells expressing TfRα or a cell surface antigen, such as EGFR, or both, but immunoprecipitation, immunocytological or immunohistochemical staining, immunofluorescence analysis and the like are preferred.

In immunoprecipitation, a cell or other agent expressing a TfR and/or a cell surface antigen, such as EGFR, is reacted with a bispecific antibody or bispecific antibody fragment of the invention, followed by the addition of a carrier that specifically binds immunoglobulins, such as protein G-Sepharose, causing the antigen-antibody complex to precipitate.

In another embodiment of the method, a bispecific antibody or bispecific antibody fragment of the present invention is immobilized on a 96-well ELISA plate and the plate is blocked with BSA-PBS. The BSA-PBS is then decanted, the plate is washed with PBS and reacted with a solution of cell or tissue lysate that expresses TfR and/or a cell surface antigen, such as EGFR. After antigen-antibody interaction, the plate is washed thoroughly and the immunoprecipitate is extracted with SDS-PAGE sample buffer. Detection or quantitation is carried out by Western blotting as described above.

In immunocytological or immunohistochemical staining, cells or tissue expressing the desired antigen or the like are treated with a surfactant or methanol or the like to increase the permeability of the cell membrane to antibodies, and reacted with a bispecific antibody of the present invention, followed by anti -immunoglobulin antibody or binding fragment thereof, labeled with a fluorescent label, for example FITC or the like, or an enzyme, for example peroxidase, or biotin, etc.; the mark is then visualized and observed using a microscope. Detection is also possible by immunofluorescence analysis, in which a fluorescently labeled antibody interacts with the cells, and the analysis is carried out using a flow cytometer [Monoclonal Antibodies - Principles and Practice, 3rd edition, Academic Press (1996), Monoclonal Antibody Experimental Manual, Kodansha scientific books (1987)]. In particular, the bispecific antibodies or bispecific antibody fragments of the present invention allow the detection of TfR and/or cell surface antigen, for example EGFR, expressed on the cell membrane by immunofluorescence analysis.

By using the FMAT 8100 HTS (manufactured by Applied Biosystems, Inc.) or the like to fluorescently label antibodies, the amount of antigen or antibody can be measured without the need to separate the antigen-antibody complex from free antibody or antigen molecules not involved in the formation of the antigen-antibody complex. antibody.

Examples

Hereinafter, this invention is described specifically by way of examples, which, however, do not limit the scope of the invention.

Example 1. Preparation of soluble human and simian TfR and EGFR antigens

1. Obtaining soluble antigens

human and simian TfR

Proteins were prepared that were extracellular domains of human and simian TfR, in which FLAG-Fc was attached to the N-terminus, as described below. The nucleotide sequence encoding the extracellular domain of human TfR is represented by SEQ ID NO: 1; the amino acid sequence deduced from it is represented by SEQ ID NO: 2; the nucleotide sequence encoding the extracellular domain of simian TfR is represented by SEQ ID NO: 3; the amino acid sequence deduced from it is SEQ ID NO: 4.

(1) Preparation of vectors for FLAG-Fc-human TfR and FLAG-Fc-simian TfR

A fragment of the human TfR extracellular domain gene was synthesized, consisting of the nucleotide sequence represented by SEQ ID NO: 1, based on the nucleotide sequence of the human TfR gene (GenBank accession number NM_003234.2, SEQ ID NO: 5; the amino acid sequence encoded by this nucleotide sequence, represented by SEQ ID NO: 6).

Vector INPEP4 (manufactured by Biogen-IDEC GmbH) containing FLAG tag sequences and human IgG Fc regions was digested with restriction enzymes BamHI and KpnI, and the nucleotide sequence represented by SEQ ID NO: 1 was inserted into the vector; thus obtaining an expression vector for the formation of FLAG-Fc-human TfR.

In the same way, an expression vector for the formation of FLAG-Fc simian TfR was obtained, containing a fragment of the simian Tfr extracellular domain gene, consisting of the nucleotide sequence represented by SEQ ID NO: 3, based on the nucleotide sequence of the simian TfR gene (SEQ ID NO: 7; amino acid the sequence encoded by this nucleotide sequence is represented by SEQ ID NO: 8).

(2) Preparation of FLAG-Fc-human TfR and FLAG-Fc-monkey TfR proteins

The expression vector encoding FLAG-Fc-human TfR obtained in 1-(1) was introduced into human embryonic kidney line HEK 293 cells using the FreeStyle (trademark) 293 expression system (manufactured by Thermo Fisher, Inc.) and the cells were cultured. so that transient expression of the specified protein occurs. 5 days after the introduction of the vector into the cells, the cell culture supernatant was collected and filtered through a membrane filter (manufactured by Millipore Corporation) with pores with a diameter of 0.22 μm.

The culture supernatant was affinity purified on Protein A resin (MabSelect, manufactured by GE Healthcare, Inc.). Antibodies adsorbed to protein A were washed with Dulbecco's phosphate buffered saline solution [liquid, without calcium and magnesium; hereinafter referred to as D-PBS(-), manufactured by Nacalai Tesque, Inc.], eluted with sodium citrate buffer solution (100 mM; pH 3.5) and collected in a tube containing Tris-HCl solution (2 M; pH 8.5 ).

The latter buffer solution was then changed to D-PBS(-) by ultrafiltration using VIVASPIN (manufactured by Sartoryus), followed by filtration sterilization using a Millex-Gv membrane filter (manufactured by Millipore Corporation) with a pore diameter of 0.22 μm; resulting in the FLAG-Fc-human TfR protein. The FLAG-Fc-monkey TfR protein was prepared in the same way. using an expression vector encoding FLAG-Fc-simian TfR, which was obtained from 1-(1). The concentration of the resulting proteins was determined by measuring the absorption at a wavelength of 280 nm and using in the calculations the extinction coefficient based on the amino acid sequence of the protein.

2. Preparation of soluble human and simian EGFR antigens

Proteins were prepared that were the extracellular domains of human and simian EGFR, in which FLAG-Fc GST was attached to the C-terminus as described below. The nucleotide sequence encoding the signal amino acid sequence and extracellular domain of human EGFR is shown in SEQ ID NO: 9; the amino acid sequence deduced therefrom is represented by SEQ ID NO: 10. The nucleotide sequence encoding the signal amino acid sequence and extracellular domain of simian EGFR is represented by SEQ ID NO: 11; the amino acid sequence deduced from it is represented by SEQ ID NO:12.

A fragment of the human EGFR extracellular domain gene was synthesized, consisting of the nucleotide sequence represented by SEQ ID NO: 9, based on the nucleotide sequence of the human EGFR gene (GenBank accession number NM_005228.4, SEQ ID NO: 13; the amino acid sequence encoded by this nucleotide sequence, represented by SEQ ID NO: 14). In the same way, a fragment of the gene for the extracellular domain of simian EGFR was synthesized, consisting of the nucleotide sequence represented by SEQ ID NO: 11. The proteins of human EGFR-FLAG-Fc, simian EGFR-FLAG-Fc, human EGFR-GST, simian EGFR-GST were obtained as described in points 1-(1) and (2).

The concentration of the resulting proteins was determined by measuring the absorption at a wavelength of 280 nm and using in the calculations the extinction coefficient based on the amino acid sequence of the protein.

Example 2: Preparation of CHO cells for expression

human or simian TfR on the cell membrane

The human TfR gene represented by SEQ ID NO: 5 was cloned into the pKANTEX vector (described in US Pat. No. 6,423,511) to thereby obtain the pKANTEX-human TfR (complete) expression vector for expressing human TfR on the cell membrane.

The resulting expression vector pKANTEX-human TfR (full) was introduced into CHO cells and the cells were cultured to transiently express the corresponding protein. After gene introduction, individual single cells were cultured to obtain colonies, thereby obtaining human TfR/CHO cells.

In the same way, the simian TfR gene represented by SEQ ID NO: 7 was cloned, and simian TfR/CHO cells were obtained.

Cells stained with the intracellular fluorescent dye 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) were prepared as follows. Human TfR/CHO and monkey TfR/CHO cells were treated with 0.02% EDTA-PBS (Nacalai Tesque, Inc.), harvested, and supplemented with CFSE (Sigma-Aldrich Co. LLC) to a final concentration of 0.2 μM; then allowed to react at room temperature for 10 minutes, after which a solution of D-PBS(-) containing 10% fetal bovine serum (Nacalai Tesque, Inc.) was added to the cells and the cells were incubated at 37°C for 10 minutes. After the reaction, the cells were centrifuged and washed with 1% BSA-PBS (Nacalai Tesque, Inc.) containing 0.02% EDTA and 0.05% NaN ₃ . The resulting CFSE-stained cells were frozen in CELLBANKER 1 medium (Nippon Zenyaku Kogyo Co., Ltd.).

Example 3. Immunization of animals for production

human antibodies

To obtain monoclonal antibodies to human TfR and human EGFR, animals that can produce human antibodies were immunized with FLAG-Fc-human TfR or human EGFR-FLAG-Fc adjuvanted for a total of 4-5 times. Serum was collected from immunized animals and binding activity to human TfR or human EGFR was determined by ELISA to confirm the increase in antibody titer.

Example 4. Preparation of antibodies to TfR

1. Preparation of phage M13 library of human antibodies with A27 light chain after immunization with TfR

RNA was extracted from spleen cells or peripheral blood mononuclear cells (PBMC) obtained from immunized animals using the RNeasy Mini kit (manufactured by QIAGEN, Inc.). The cDNA was amplified using the SMARTer RACE cDNA amplification kit (manufactured by Clontech Laboratories, Inc.), and then the VH gene fragment was amplified by PCR. The VH gene fragment and the nucleotide sequence of A27, which is the germline sequence of a human antibody and consists of the nucleotide sequence represented by SEQ ID NO: 15, was inserted into the phagemid vector pCANTAB 5E (manufactured by Amersham Pharmacia, Inc.), which was transformed into E. coli TG1 cells (manufactured by Lucigen Corporation), thereby obtaining plasmids.

Note that the nucleotide sequence of A27 encodes a human antibody VL consisting of the amino acid sequence represented by SEQ ID NO: 16; the amino acid sequences of CDRs 1, 2 and 3 of this VL (designated LCDR 1, 2 and 3, respectively) are represented by SEQ ID NOS: 17, 18 and 19, respectively.

By infecting the interference-resistant helper phage VCSM13 (manufactured by Agilent Technologies, Inc.), the resulting plasmids produced an M13 phage library of human antibody generated by TfR immunization, in which the VL gene consists of the nucleotide sequence A27 and which includes the VH gene library.

2. Preparation of monoclonal antibody to TfR

with light chain A27

Using an M13 phage library of human antibody generated by TfR immunization, a monoclonal anti-TfR antibody containing VL encoded by the nucleotide sequence A27 was produced by phage display.

(1) Protein panning with immobilization on the walls of the tube.

Human TfR (manufactured by BBI Solutions, Inc.) was immobilized on the surface of MAXISORP STAR TUBE tubes (manufactured by NUNC, Inc.) and the portion to which human TfR did not bind was blocked with SuperBlock Blockig Buffer reagent (manufactured by Thermo Fisher, Inc.); the M13 phage library of human antibodies was introduced, the reaction proceeded at room temperature for 1-2 hours; then washed 3 times with D-PBS(-) and PBS containing 0.1% Tween 20 (hereinafter referred to as PBS-T; manufactured by Wako Pure Chemical Industries, Ltd.), after which the bound phage particles were eluted with glycine-buffered solution. HCl (0.1 M; pH 2.2); the eluate was neutralized with Tris-HCl buffer solution (2M; pH 8.5).

(2) Panning of CHO cells, on the membrane of which human or simian TfR is expressed

The human M13 phage library was incubated with CHO cells on ice for 30 minutes; centrifuged to separate the cells and collect the supernatant, thus obtaining a solution with phage particles interacting with CHO cells. The phage solution was then incubated with human TfR/CHO stained with CFSE, which was prepared according to Example 1, on ice for 30 minutes to 1 hour, after which it was centrifuged and the supernatant was discarded. Wash several times with D-PBS(-) solution containing 5% fetal bovine serum, 1 mM EDTA and 0.1% NaN ₃ (hereinafter referred to as SM buffer solution), after which primary antibodies, anti-M13 antibodies (GE Healthcare) were added , Inc.) and allowed to react on ice for 30 minutes. Centrifuged, the supernatant was discarded, the pellet was washed several times with SM buffer solution, after which secondary antibodies were added - goat anti-mouse IgG (H+L) labeled with allophycocyanin (APC; Southern Biotech, Inc.) and allowed to react on ice for 30 minutes. Centrifuge, discard the supernatant, wash the pellet several times with SM buffer, then add 7-aminoactinomycin D (AAD; BD Biosciences, Inc.) and allow to react for on ice for 30 minutes.Viable cells that did not stain with 7-ADD and carried antigen that stained with CFSE were collected using a flow cytometer (BD Biosciences, Inc., FACS Aria III). In addition, when phage particles were confirmed to bind to cells expressing a given antigen, cells that bound phage and stained with APC were also selected. Phage particles bound to the selected cells were eluted with a glycine-HCl buffer solution (0.1 M; pH 2.2); the eluate was neutralized with Tris-HCl buffer solution (2 M; pH 8.5).

Cell panning where simian TfR/CHO cells were used was carried out in a similar manner. The eluted phage was infected with competent E. coli TG1 cells for its amplification. The steps described in (1) or (2) above were repeated 2-3 times to concentrate phage exhibiting scFvs that specifically bind to human TfR. The concentrated phage was used to infect E. coli TG1 cells, which were then plated on plates containing SOBAG medium (2.0% tryptone; 0.5% yeast extract; 0.05% NaCl; 2.0% glucose; 10 mM MgCl ₂ ; 100 μg/ml ampicillin; 1.5% agar) to form colonies.

Colonies were transferred to fresh medium, cultured and infected with interference-resistant helper phage VCSM13 and cultured again; in this way a monoclonal phage was obtained. Using the resulting monoclonal phage, a clone binding to both human and simian TfR was selected using a flow cytometer (Millipore Corporation, Guava easy Cyte HT).

For flow cytometry, the phage clone was added to human TfR/CHO or monkey TfR/CHO cells according to Example 2. Incubated at 4°C for 30 minutes to perform the reaction, after which each well was washed 3 times with PBS-T solution.

ELISA was performed to evaluate the binding activity of the monoclonal phage to TfR. Namely, a phage solution diluted with PBS-T containing 10% Block Ace blocking reagent (manufactured by Dainippon Pharmaceutical Co., Ltd.) was added to the wells of a 96-well immunological plate (NUNC, Inc.) with immobilized antigen protein and incubated at room temperature for 1 hour to perform the reaction, after which they were washed with PBS-T solution and horseradish peroxidase-labeled anti-M13 antibodies (manufactured by GE Healthcare, Inc.) diluted in 1% BSA-PBS were added and incubated at room temperature. temperature for 1 hour to carry out the reaction. The plate was then washed with PBS-T, TMB chromogenic substrate solution (manufactured by DAKO, Inc.) was added and left at room temperature for the color reaction to occur, which was stopped by adding hydrochloric acid (2N); absorbance was measured at 450 nm (reference wavelength 570 nm) using a plate reader (Emax, Molecular Devices, Inc.).

Sequences were analyzed for clones that bound both human and simian TfR; In this way, antibodies to TfR were obtained, in which VL was encoded by the nucleotide sequence A27. Table 1 shows the numbers (SEQ ID NO) of the complete nucleotide sequences encoding the VH of each of the obtained antibodies to TfR, the amino acid sequences derived from them and the amino acid sequences CDR 1-3 VH (hereinafter sometimes also referred to as HCDR 1-3). The “-” icon means that there is no information for this sequence.

Table 1. Variable region sequence numbers

heavy chain (VH) anti-TfR antibodies

Clone PSM4072 R327 TfR1071 TfR4016 Cyno186 Cyno292 TfR1007 Cyno163 T14 Nucleotide
VH sequence 20 25 thirty 35 40 45 - - - Amino acid sequence of VH 21 26 31 36 41 46 50 51 52 Amino acid sequence of HCDR1 22 27 32 37 42 47 107 - - Amino acid sequence of HCDR2 23 28 33 38 43 48 108 - - Amino acid sequence of HCDR3 24 29 34 39 44 49 109 - -

3. Obtaining antibodies to TfR,

in which the light chain is other than A27

(1) Production of hybridoma by cell fusion

Murine myeloma cell line P3-U1 (P3X63Ag8U.1, ATCC accession number CRL-1597) was cultured in S-Clone medium (EIDIA Co., Ltd.), adapted to serum-free conditions, and then used as the parental line for cell fusion . The spleen was aseptically removed from immunized animals and hemolysed using RED BLOOD CELL LYSING BUFFER (Sigma-Aldrich Co. LLC); the resulting cells were washed twice with PBS and mixed with P3-U1 cells in the ratio (spleen cells): (P3-U1) = 8:1; the cell mixture was centrifuged at 1200 rpm for 5 minutes.

The resulting cell clusters in the resulting precipitate were thoroughly loosened, and 0.5 ml of a mixed solution of 1 g of polyethylene glycol-1000 (PEG-1000, Junsei Chemical Co., Ltd.), 1 ml of MEM medium (Nacalai Tesque, Inc.) and 0. 35 ml of dimethyl sulfoxide (Sigma-Aldrich Co. LLC) at 37°C, then MEM medium was added - first 1 ml 5 times at 1 min intervals, then to a total volume of 50 ml.

The cell suspension was centrifuged at 900 rpm for 5 minutes, and the cells in the resulting pellet were gently loosened and the spleen cells were suspended at a density of 1.5 x 10 ⁷ cells/9 ml in S-Clone medium containing HAT SUPPLEMENT; (Thermo Fisher Scientific, Inc.). 100 μl of S-Clone medium with the addition of HAT was added to the wells of a 96-well plate; 100 μl of cell suspension was added to the wells prepared in this way and cultured in a CO ₂ incubator (5% CO ₂ ) at a temperature of 37°C for 8-10 days.

(2) Hybridoma screening

The TfR binding activity of antibodies contained in the hybridoma culture supernatant was determined by ELISA. Samples were taken from the supernatant of the hybridoma culture, measurements were carried out as described above in section 2.(2).

(3) Hybridoma subcloning

Cells from screening-positive wells were subcloned and cultured in S-Clone cloning medium for 7-10 days.

(4) Cloning and sequencing of the antibody variable region gene

Total RNA was isolated from cloned hybridomas using the RNeasy Mini kit (QIAGEN, Inc.) and antibody genes were amplified by the 5’-RACE method. The SMARTer RACE Kit (Clontech Laboratories, Inc.) was used to synthesize cDNA for RACE. The amplified antibody variable region sequence was cloned using the TOPO TA Cloning kit (Invitrogen, Inc.) to confirm the sequence of the desired DNA fragment.

From among the anti-TfR antibodies obtained, the amino acid sequences deduced from the nucleotide sequences encoding the variable regions of the heavy and light chains of clone 2230 are represented by SEQ ID NO: 53 and SEQ ID NO: 54, respectively.

4. Expression vector construction

obtained antibodies to TfR

Vectors were obtained for the expression of soluble IgG with the genes of the resulting antibodies to TfR built into them. First, the A27 gene encoding the common VL was subcloned into the N5KG4PE vector (IDEC Pharmaceuticals) or N5KG4PE R409K (described in WO 2006/033386).

The VH gene was then subcloned into N5KG4PE or N5KG4PE R409K; In this way, expression vectors were obtained for the production of monoclonal antibodies to TfR with the human IgG4PE constant region or with the human IgG4PE R409K constant region.

To use as a positive control the TfR monoclonal antibodies TfR434 and TfR435, which are TfR neutralizing antibodies (described in WO 2012/153707), appropriate expression vectors were prepared. The VH amino acid sequences of antibodies TfR434 and TfR435 are represented by SEQ ID NOS: 55 and 56, respectively, the VL amino acid sequence is presented by SEQ ID NO: 57.

The genes encoding the VH and VL antibodies TfR435 were synthesized and subcloned into the N5KG1 vector (described in WO 2003/033538); thus, an expression vector for the anti-TfR monoclonal antibody TfR435 with the human IgG1 constant region was obtained.

Hereinafter, all these monoclonal antibodies are referred to by their respective clone designations.

Example 5. Preparation of antibodies to cell surface antigen

I. Obtaining antibodies to EGFR

1. 1. Preparation of human M13 phage antibody library after EGFR immunization

An M13 human antibody phage library, after immunization with EGFR with the VL gene consisting of the nucleotide sequence A27, or with the VL gene of the anti-TfR 2230 antibody, including the VH gene library, was prepared in the same way as described in Example 4, 1.

2. Obtaining antibodies to EGFR

Human or simian EGFR-Fc protein obtained in Example 1 was immobilized on MAXISORP STARTUBE tubes (manufactured by NUNC, Inc.), and the part to which the antigen did not bind was blocked with SuperBlock Blockig Buffer reagent (manufactured by Thermo Fisher, Inc.); After immunization with EGFR, a library of human antibodies with phage M13 exhibiting scFv that specifically binds to human EGFR and simian EGFR was prepared and monocloned as described in Example 4, 2.

These steps were repeated 3-5 times to concentrate phage exhibiting scFvs that specifically bind to human EGFR-Fc and simian EGFR-Fc.

Confirmation that the clone specifically recognizes human EGFR-Fc and monkey EGFR-Fc was performed by ELISA, which was performed as described in Example 4, with immobilization of human EGFR-Fc or monkey EGFR-Fc in Example 1.

From among the clones that bound to both human and simian EGFR, three clones were selected with high binding affinities comparable to those of cetuximab. For all three clones, sequence analysis was performed and antibodies to EGFR were obtained with VL encoded by the nucleotide sequence A27. Information on the VH of these antibodies to EGFR is presented in Table 2 according to the same scheme as in Table 1.

Table 2. VH sequences of antibodies to EGFR

E08 E12 E17 Nucleotide sequence,
encoding VH SEQ ID NO: 58 SEQ ID NO: 63 SEQ ID NO: 68 Amino acid sequence of VH SEQ ID NO: 59 SEQ ID NO: 64 SEQ ID NO: 69 Amino acid sequence
HCDR1 SEQ ID NO: 60 SEQ ID NO: 65 SEQ ID NO: 70 Amino acid sequence
HCDR2 SEQ ID NO: 61 SEQ ID NO: 66 SEQ ID NO: 71 Amino acid sequence
HCDR3 SEQ ID NO: 62 SEQ ID NO: 67 SEQ ID NO: 72

Similarly, antibodies to EGFR were obtained with the same VL as for antibodies to TfR 2230. From the number of antibodies obtained to EGFR for clones KME07, KME09 and KME11, amino acid sequences were deduced from the nucleotide sequences encoding the variable regions of the heavy chains, which are represented by SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75 respectively.

Vectors for the expression of soluble IgG with genes encoding antibodies to EGFR built into them were obtained in the same way as described in Example 4. The constant region of human antibodies IgG4PE or IgG4PE R409K was used as the constant region.

As a positive control for anti-EGFR antibodies, the variable region antibody of the anti-EGFR monoclonal antibody C225 described in WO 1996/040210 (hereinafter referred to as cetuximab) was prepared. The amino acid sequence of cetuximab VH is represented by SEQ ID NO: 76. The amino acid sequence of cetuximab VL is represented by SEQ ID NO: 77.

The genes encoding VH and VL of cetuximab were synthesized and subcloned into the N5KG1 vector as described in Example 4; Thus, an expression vector for cetuximab with the human IgG1 constant region was obtained.

II. Obtaining antibodies against Glypican-3

An antibody with the variable region of the anti-glypican-3 monoclonal antibody HN3 described in WO 2012/145469 was prepared. The amino acid sequence of the HN3 VH antibody is shown by SEQ ID NO: 78. The gene encoding the HN3 VH was synthesized and subcloned into the N5KG4PE R409K vector as described in Example 4; thus, the anti-glypican-3 monoclonal antibody HN3 with the constant region of human IgG4PE R409K was prepared.

Example 6: Construction of an expression vector to produce a bispecific antibody that binds to TfR

and with cell surface antigen

A bispecific antibody having the structure shown in FIG. 1(A), and binding to human and simian TfR, as well as human and simian EGFR, were prepared as follows. In fig. 1(A) - 1(C) in the illustrated antibodies, the VH as part of the N-terminal side of the polypeptide is designated VH1, and the VH as part of the IgG portion of the C-terminal side is designated VH2. These bispecific antibodies have the form described in WO 20 9/131239.

The VH1 and VH2 regions are the VH of the antibody prepared in Example 4 or 5, and VH1 and VH2 are the VH of antibodies against different antigens.

Hereinafter, in the present description, in cases where an N-terminal side polypeptide and an IgG isotype antibody are connected via a linker, the gene encoding the amino acid sequence of the linker is called a linker gene.

The bispecific antibodies prepared as described below used an amino acid sequence with part of the IgG4 hinge region as a linker [ES (SEQ ID NO: 79), ESKYG (SEQ ID NO: 80), ESKYGPP (SEQ ID NO: 81), etc. .], or a GS type linker (GGGGS (SEQ ID NO: 82)], or a sequence of three repeats GGGGS). As the heavy chain constant region of the IgG part, the heavy chain constant region IgG4PE or IgG4PE R409K (the nucleotide sequence encoding it is represented by SEQ ID NO: 83, the corresponding amino acid sequence is represented by SEQ ID NO: 84) described in WO 2006/033386 was used.

Hereinafter, a bispecific antibody in which the N-terminal side polypeptide includes the variable region of an anti-EGFR antibody and the IgG portion includes the variable region of an anti-TfR antibody is referred to as an EGFR-TfR bispecific antibody. Also, for example, an EGFR-TfR bispecific antibody including the variable region of the anti-EGFR antibody E12 and the variable region of the anti-TfR antibody TfR1071 is called an E12-TfR1071 bispecific antibody. Likewise, a bispecific antibody in which the N-terminal side polypeptide includes the variable region of an anti-TfR antibody and the IgG portion includes the variable region of an anti-EGFR antibody is called a TfR-EGFR bispecific antibody.

Also, for example, a TfR-EGFR bispecific antibody including a variable region of an anti-TfR antibody TfR1071 and a variable region of an anti-EGFR antibody E12 is called a TfR1071-E12 bispecific antibody. An antibody that has a linker between the N-terminal side polypeptide and the IgG portion is called an EGFR linker-TfR bispecific antibody.

Also, for example, an EGFR linker-TfR bispecific antibody including an anti-EGFR antibody E08 variable region, an anti-TfR antibody TfR1071 variable region, and an ES linker is called an E08-ES-TfR1071 bispecific antibody. Note that if the linker is GS (SEQ ID NO: 82), then in the name of the bispecific antibody it is designated GS, and if the linker is a sequence in which the GS linker is repeated 3 times, then in the name of the bispecific antibody it is designated GS3.

The designations, structures, and linkers of the bispecific antibodies of this invention, the designations of the anti-TfR antibody clones used to produce the bispecific antibodies, and the designations of the antibody clones to the cell surface antigen are given in Table 3. Note that in this table, the sign “-” indicates that the linker is missing and the corresponding sequence is not given.

Table 3

Designation
bispecific antibody Structure VH1 Linker VH2 E08-PSM4072 Fig. 1(A) E08 - PSM4072 E08-R327 Fig. 1(A) E08 - R327 E08-TfR1071 Fig. 1(A) E08 - TfR1071 E08-TfR4016 Fig. 1(A) E08 - TfR4016 E08-cyno186 Fig. 1(A) E08 - cyno186 E08-cyno292 Fig. 1(A) E08 - cyno292 E08-T14 Fig. 1(A) E08 - T14 E08-ES-TfR1071 Fig. 1(A) E08 ES TfR1071 E08-ES-cyno186 Fig. 1(A) E08 ES cyno186 E08-ESKYG-TfR1071 Fig. 1(A) E08 ESKYG TfR1071 E08-ESKYG-cyno186 Fig. 1(A) E08 ESKYG cyno186 E08-ESKYGPP-TfR1071 Fig. 1(A) E08 ESKYGPP TfR1071 E08-ESKYGPP-cyno186 Fig. 1(A) E08 ESKYGPP cyno186 PSM4072-E08 Fig. 1(A) PSM4072 - E08 R327-E08 Fig. 1(A) R327 - E08 TfR1071-E08 Fig. 1(A) TfR1071 - E08 TfR4016-E08 Fig. 1(A) TfR4016 - E08 cyno186-E08 Fig. 1(A) cyno186 - E08 cyno292-E08 Fig. 1(A) cyno292 - E08 T14-E08 Fig. 1(A) T14 - E08 E12-PSM4072 Fig. 1(A) E12 - PSM4072 E12-TfR1007 Fig. 1(A) E12 - TfR1007 E12-cyno163 Fig. 1(A) E12 - cyno163 E12-R327 Fig. 1(A) E12 - R327 E12-TfR1071 Fig. 1(A) E12 - TfR1071 E12-TfR4016 Fig. 1(A) E12 - TfR4016 E12-cyno186 Fig. 1(A) E12 - cyno186 E12-cyno292 Fig. 1(A) E12 - cyno292 E12-T14 Fig. 1(A) E12 - T14 KME07-2230 Fig. 1(A) KME07 - 2230 KME09-2230 Fig. 1(A) KME09 - 2230 KME11-2230 Fig. 1(A) KME11 - 2230 HN3-TfR1071 Fig. 1(B) HN3 - TfR1071 HN3-GS-TfR1071 Fig. 1(B) HN3 G.S. TfR1071 HN3-GS3-TfR1071 Fig. 1(B) HN3 GS3 TfR1071

1. Preparation of an expression vector for a bispecific antibody

with general light chain

An expression vector for the general type light chain bispecific antibody shown in FIG. 1(A), from among the bispecific antibodies presented in Table 3, were prepared as described below.

First, the A27 nucleotide sequence encoding the common VL was subcloned into the N5KG4PE R409K vector (described in WO 2006/033386).

Then, taking as a template the gene (SEQ ID NO: 85), encoding the same amino acid sequence as the CH1 region of human IgG4, with changed codons, this gene fragment was amplified by PCR using the KOD-Plus-Ver.2 reagent (Toyobo Co., Ltd.). Using the VH gene of the antibody obtained in Example 4 or 5 as a template, a fragment of the nucleotide sequence encoding VH2 was amplified in the same way.

The resulting two fragments were subcloned into the antibody expression vector of Example 4 or 5 between the sequences encoding the VH1 and CH1 portions of IgG; in this way an expression vector for the desired bispecific antibody was obtained.

2. Preparation of an expression vector for a bispecific antibody

with light chain, non-general type

An expression vector for a non-generic light chain bispecific antibody shown in FIG. 1(A) from among the bispecific antibodies presented in Table 3 was prepared as described below.

The antibody shown in FIG. 1(B), is formed using a specific type of expression vector. First, the A27 nucleotide sequence encoding VL was subcloned into the pCI-OtCAG-G4PE R409 vector containing the nucleotide sequence encoding the human IgG4PE R409K constant region.

This vector is designated pCI-A27. The constant region encoded by the nucleotide sequence within the vector pCI-OtCAG_hG4PE R409K is the constant region of the heavy chain of mutant IgG4 [hereinafter referred to as IgG4PE R409K (WO 2006/033386)], obtained by replacing Ser at position 228 with Pro, Leu in position 235 on Glu and Arg at position 409 on Lys (EU numbering) in the amino acid sequence of the human IgG4 heavy chain constant region.

Note that such a vector is prepared by total synthesis with the introduction of restriction sites necessary for expression of the human antibody gene using the pCI vector (Promega Corporation) as a backbone.

After the nucleotide sequences encoding the VH and the linker antibody of Example 5 were inserted, the completely synthesized nucleotide sequence in which the nucleotide sequence encoding the linker and the VH gene of the anti-TfR antibody of Example 4 were ligated was subcloned into the pCI-A27 vector; in this way an expression vector for the desired antibody was obtained.

Example 7: Preparation of a monoclonal antibody that binds

with TfR, a monoclonal antibody that binds

with a cell surface antigen, and a bispecific antibody

Expression and purification of a TfR-binding monoclonal antibody, a cell surface antigen-binding monoclonal antibody, and a bispecific antibody containing binding domains to both antigens obtained using the antibody expression vectors of Examples 4-6 were carried out as follows.

The antibody expression vector was co-transfected into Expi293 cells using the Expi293 (trade name) Expression System kit (Thermo Fisher, Inc.), and a transfection enhancer was added after 12-16 hours; thus ensuring transient expression of the antibody or bispecific.

4-7 days after vector administration, the culture supernatant was collected and filtered through a 0.22 μm membrane filter (manufactured by Millipore Corporation), followed by affinity purification of the antibody using Protein A resin (MabSelect SuRe, GE Healthcare, Inc. .). Wash with D-PBS(-) solution. The target antibody adsorbed onto Protein A resin was eluted with sodium citrate buffer (100 mM; pH 3.5) and collected in tubes containing Tris-HCl buffer (2 M; pH 8.5).

The resulting protein solution was concentrated using VIVASPIN (Sartorius), and the buffer solution was replaced with D-PBS(-) using a Nap column (manufactured by GE Healthcare, Inc.). In that case. when the purified component was monomeric, the monomeric fraction was isolated from the antibody or bispecific antibody solution using an AKTA FPLC chromatography system (manufactured by GE Healthcare, Inc.) and a Superdex high performance purification column (manufactured by GE Healthcare, Inc.). Then sterilized by filtration in an AKTA system (Millex-Gv, manufactured by Millipore Corporation) on a membrane filter with pores with a pore diameter of 0.22 μm; thus obtaining a purified antibody or a purified bispecific antibody.

In the thus obtained solution of purified antibody or purified bispecific antibody, the absorbance at a wavelength of 280 nm was measured and the concentration of the purified antibody was determined using the extinction coefficient calculated from the amino acid sequence of the antibody or bispecific antibody.

Example 8: Determination of Bispecific Binding Affinity

antibodies with antigen using the Biacore biosensor system

To determine the binding activity and cross-species cross-reactivity of EGFR-TfR-bispecific antibodies obtained in Example 7 to each of human TfR, simian TfR, human EGFR, simian EGFR, the FLAG-Fc-human TfR, FLAG-Fc proteins obtained in Example 1 were used -monkey TfR, human EGFR-GST, monkey EGFR-GST and affinity was determined by SPR using a Biacore T100 optical biosensor system (manufactured by GE Healthcare, Inc.).

Anti-human IgG antibodies were immobilized onto a CM5 sensor chip (manufactured by GE Healthcare, Inc.) using the Human Antibody Capture Kit (manufactured by GE Healthcare, Inc.) according to the accompanying instructions. The test bispecific antibody was added to the flow cell at a concentration of 1-3 μg/ml for 10 seconds at a flow rate of 10 μl/min.

Then, as an analyte, a solution of each antigenic protein in HBS-EP+ (manufactured by GE Healthcare, Inc.) containing 0.1% BSA in five three-fold serial dilutions from 1 nM to 9 nM was added at a flow rate of 30 μl/min and the association was recorded bispecific antibody and analyte within 2 minutes, dissociation within 10 minutes.

The measurements were carried out in single-cycle kinetics mode. Sensograms were analyzed using Bia Evaluation Software (manufactured by GE Healthcare, Inc.) and the reaction rate constant was calculated for each antibody.

The calculated values of the dissociation constant [kd/ka = K _D ] of each of the bispecific antibodies when interacting with human and monkey TfR and EGFR, as well as the ratio of this constant for binding to human protein to the value for binding to monkey protein are given in Table 4 The designation N/A indicates that measurements were not taken in this case.

Table 4

human EGFR
(nM) simian EGFR (nM) human EGFR (nM) /
simian EGFR (nM) human TfR
(nM) monkey TfR (nM) human TfR (nM)/
monkey TfR (nM) E08-TfR1071 0.13 0.15 0.87 5.79 12.0 0.48 E08-cyno186 N/A N/A N/A 1.56 1.97 0.79 E12-TfR1071 0.21 0.25 0.84 2.42 22.2 0.11 E12-cyno186 N/A N/A N/A 10.7 56.5 0.19 E17-TfR1071 0.31 0.67 0.46 4.09 26.6 0.15 E17-cyno186 N/A N/A N/A 4.02 5.39 0.75

As can be seen from Table 4, the K _D values for the binding of EGFR-TfR bispecific antibodies to human and simian TfR are of the order of 10 ^{-8 -} 10 ^-9 M. The K _D values for the binding of EGFR-TfR bispecific antibodies to simian TfR are 0 ,1-10 times higher than the K _D values for the binding of EGFR-TfR-bispecific antibodies to human TfR; therefore, these antibodies are cross-reactive with human TfR and simian TfR.

Similarly, K _D values for binding of EGFR-TfR bispecific antibodies to human and simian EGFR receptors are on the order of 10 ^-9 -10 ^-10 M. K _D values for binding of EGFR-TfR bispecific antibodies to simian EGFR are 0.1-10 times higher than the K _D values for the binding of EGFR-TfR bispecific antibodies to human EGFR; therefore, these antibodies are cross-reactive against human EGFR and simian EGFR.

Example 9. Determination of the level of antigen expression in cancer cells by flow cytometry and assessment of the binding affinity of the resulting antibodies

The expression levels of various antigens in different cancer cell lines were determined by fluorescence-activated cell sorting (FACS) as follows. The method used antibodies to human CD71 labeled with phycoerythrin (PE; BD Pharmingen, Inc.) and antibodies to EGFR labeled with Alexa Fluor 488 fluorescent dye (Santa Cruz Biotechnology, Inc.).

Determination of the expression level of TfR and EGFR in cancer cells of various lines was carried out as follows.

OE21 cells were suspended in staining buffer (SB), which is a D-PBS(-) solution containing 0.1% _NaN3 and 1% FBS, to a density of 1 × 10 ⁶ cells per 1 ml; this suspension was added in 100 µl into the wells of a 96-well round-bottom plate (manufactured by Falcon, Inc.) Centrifuged at 2000 rpm for 2 minutes at 4°C, the supernatant was discarded, and PE-labeled anti-human CD71 antibodies were added to the resulting pellet. or anti-EGFR antibodies labeled with Alexa Fluor 488 and incubated for 30 minutes in an ice bath.The cells were washed 2 times with SB solution, suspended in the same solution, and the fluorescence intensity in each well was measured using a FACSCANTO II cytometer (manufactured by BD Biosciences, Inc. .) The level of TfR expression in cells of the T.Tn, U-937, HepG2, HuH-7 and HLE lines was also determined.

The expression level of GPC3 in HepG2, HuH-7 and HLE cells was determined similarly. Namely, cells were prepared as described above, using HN3 antibodies as primary antibodies; as secondary antibodies, goat anti-human IgG (H+L) labeled with Alexa Fluor (registered trademark) 488 (Molecular Probes, Inc.), then incubated for 30 minutes in an ice bath; fluorescence intensity was measured as described above.

PBS and IgG4 R409K mutant antibodies (hereinafter referred to as anti-DNP antibodies) obtained by the method described in Example 5 of WO 2006/033386 using a vector encoding VH and VL (GenBank accession numbers U16688) were used as a negative control and U116687, respectively) antibodies to DNP-1, described in Mol. Immunol. 1996 Jun; 33 (9): 759-68.

In fig. 2(A)-2(C) show the results of flow cytometry determination of the expression level of EGFR in OE21, T.Tn and U-937 cells, respectively. In fig. 2(D)-2(F) depict the results of flow cytometry determination of the expression level of TfR in OE21, T.Tn and U-937 cells, respectively. As can be seen from these illustrations, the level of EGFR expression is high in OE21 cells, medium in T.Tn cells, and low in U-937 cells.

In fig. 3(A)-3(C) show the results of flow cytometry determination of the expression level of GPC3 in HepG2, HuH-7 and HLE cells, respectively. In fig. 3(D)-3(F) depict the results of flow cytometry determination of the expression level of TfR in HepG2, HuH-7 and HLE cells, respectively. As can be seen from these illustrations, the level of GPC3 expression in HepG2 cells is high, in HuH-7 cells it is medium, and in HLE cells this protein is not expressed. In all cells: HepG2, HuH-7 and HLE, the level of EGFR expression is low.

Judging by Fig. 2 and 3, in the studied cancer cell lines, the expression of TfR varied slightly, and the level of expression of this protein was from low to medium.

The binding affinity of anti-TfR antibodies and anti-EGFR antibodies obtained in Examples 4 and 5 to OE21 cells was confirmed by flow cytometry. In fig. 4(A)-4(I) depict the results obtained with antibodies E08, E12, E17, KME07, KME09, KME11, TfR1071, 2230 and T14, respectively.

Of all the antibodies to EGFR, binding to OE21 cells was not confirmed only in the case of KME11 antibodies. Antibody KME11 bound to the soluble EGFR protein described in Example 5, but did not bind to cell surface membrane-associated EGFR. Of all TfR antibodies, binding to OE21 cells was not confirmed only in the case of T14 antibodies. Antibody T14 bound to soluble TfR (by ELISA, see Example 4) but did not bind to membrane-associated TfR on the cell surface.

Example 10 Determination of cell growth inhibition by anti-TfR antibodies

The ability of monoclonal antibodies to TfR obtained in Example 4 to inhibit cell growth was determined as described below, taking as an indicator the suppression of proliferation of cancer cells of a certain line.

OE21 cells were seeded (1 × 10 ³ cells) into the wells of a flat-bottomed 96-well plate (manufactured by Falcon, Inc.) and the test antibody diluted in RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS was added. , to a final concentration of 1 μg/ml; incubated at 37°C in an atmosphere containing 5.0% carbon dioxide for 4-6 days. CellTiter-Glo (registered trade name) Luminescent Cell Vialbility Assay (manufactured by Promega Corporation) was then used and fluorescence intensity was measured using a microplate reader (1420 ARVO Multi-Label Counter, manufactured by WALLAC, Inc.). For each variant of conditions, 6 wells were allocated and the average value was calculated. The fluorescence intensity obtained for a given well reflects the level of ATP in viable cells in that well. Anti-DNP antibodies were used as a negative control.

Also, along with the test antibody, polyclonal anti-human IgG antibodies (Sigma, Inc.) (hereinafter referred to as cross-linking antibodies) were added at a concentration of 10 μg/ml and, under conditions where the test antibody was cross-linked, the procedures described above were performed. measurements. The obtained data are presented in Fig.5. From among the antibodies tested, those monoclonal antibodies that themselves did not exhibit the ability to inhibit cell growth were selected as antibodies that were not able to neutralize TfR.

By adding a cross-linking antibody to cross-link the two antibodies tested, clones were identified that exhibited the ability to inhibit cell growth (TfR1071, TfR4016, cyno186, cyno292 and 2230) and those that did not (PSM4072, TfR1007 and cyno163) .

Example 11 Determination of cell growth inhibition by antibodies

to cell surface antigen

The ability of monoclonal antibodies to cell surface antigen obtained in Example 5 to inhibit cell growth was determined as described below, taking as an indicator the suppression of proliferation of cancer cells of a certain line.

OE21 cells were seeded (1 × 10 ³ cells) into the wells of a flat-bottomed 96-well plate (manufactured by Falcon, Inc.) and the test antibody diluted in RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS was added. , to various concentrations; cells were cultured at 37°C in an atmosphere containing 5.0% carbon dioxide for 4-6 days. CellTiter-Glo (registered trade name) Luminescent Cell Vialbility Assay (manufactured by Promega Corporation) was then used and fluorescence intensity was measured using a microplate reader (1420 ARVO Multi-Label Counter, manufactured by WALLAC, Inc.). For each variant of conditions, 3 wells were allocated and the average value was calculated. The fluorescence intensity obtained for a given well reflects the level of ATP in viable cells in that well. Anti-DNP antibodies were used as a negative control.

OE21 cells were used to determine cell growth inhibition by anti-EGFR antibodies. The results obtained with these cells are presented in Fig. 6. As can be seen from Fig. 6, cetuximab and clone E08 had a high ability to suppress cell growth, E17 antibodies showed this ability weakly, and clone E12 did not have it at all. These results are considered to reflect the neutralizing activity of these antibodies against EGFR.

Example 12 Cell Growth Inhibition Determination

bispecific antibodies

The ability of the bispecific antibodies obtained in Example 6 to inhibit cell growth was determined as described below, taking as an indicator the suppression of proliferation of cancer cells of a certain line.

Cancer cells were seeded (1 × 10 ³ cells) into the wells of a flat-bottomed 96-well plate (manufactured by Falcon, Inc.) and the test antibody diluted in RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS was added. to various concentrations; cells were cultured at 37°C in an atmosphere containing 5.0% carbon dioxide for 4-6 days. CellTiter-Glo (registered trade name) Luminescent Cell Vialbility Assay (manufactured by Promega Corporation) was then used and fluorescence intensity was measured using a microplate reader (1420 ARVO Multi-Label Counter, manufactured by WALLAC, Inc.). For each variant of conditions, 3 wells were allocated and the average value was calculated. The fluorescence intensity obtained for a given well reflects the level of ATP in viable cells in that well. Anti-DNP antibodies were used as a negative control.

OE21 cells were used to determine cell growth inhibition by bispecific antibodies. In fig. 7A-7C show the results obtained for the anti-EGFR E08 variable region bispecific antibody, the anti-EGFR E12 variable region bispecific antibody, and the anti-TfR 2230 variable region bispecific antibody, respectively.

As can be seen from Fig. 7A, a high ability to inhibit cell growth was observed when the variable region of a clone (for example, TfR1071), which exhibited the ability to inhibit cell growth under conditions of cross-linking of antibody molecules with the cross-linking antibody of Example 10, was located on the side of the IgG part, and the variable region anti-EGFR antibody was located on the N-terminal side of the polypeptide. Moreover, the same ability to suppress cell growth was observed if a piece of the amino acid sequence of the hinge region (ES, ESKYG or ESKYGPP) of IgG4 served as a linker. And the bispecific antibody, which had the variable region of the anti-TfR antibody in the N-terminal side of the polypeptide, practically did not inhibit cell growth.

Note that, according to the results of Example 9, the T14 antibody to TfR T14 does not interact with TfR on the cell surface - therefore, the ability to suppress cell growth in antibodies E08-T14 and T14-E08 can be considered due to the suppression of the signaling pathway involving EGFR due to E08 alone ( Example 11).

As can be seen from Fig. 7B, bispecific antibodies, in which the IgG part contains a variable region of any of the clones (R327, TfR1071, TfR4016, cyno186 and cyno292), capable of inhibiting cell growth under conditions of cross-linking of antibody molecules with a cross-linking antibody according to Example 10, have greater ability to suppress cell growth than bispecific antibodies, which in the IgG part have a variable region of one of the clones (PSM4072, TfR1007 and cyno163) that are not capable of suppressing cell growth in the conditions of cross-linking of antibody molecules with a cross-linking antibody

With regard to bispecific antibodies containing the variable region of the anti-TfR antibody 2230, as can be seen from FIG. 7C, antibodies KME07-2230, KME09-2230 and KME11-2230 are capable of inhibiting cell growth. As stated in Example 9, KME11 does not bind to EGFR on cells—hence, the bispecific antibody KME11-2230 has the ability to inhibit cell growth in an EGFR-independent manner. There is reason to fear that clones with this property may be toxic to normal cells that do not have EGFR but express TfR, such as bone marrow cells.

The results described above show that bispecific antibodies containing in the IgG part the variable region of antibodies to TfR, which by themselves are not able to suppress cell growth, but exhibit this ability when cross-linked (R327, TfR1071, TfR4016, cyno186 and cyno292), have high ability to inhibit cell growth selectively to cell surface antigen. In addition, it has been shown that the ability of bispecific antibodies to inhibit cell growth is not affected by the presence or absence of a linker.

To prove the relationship between the ability of EGFR-TfR bispecific antibodies to inhibit cell growth and the level of EGFR expression, this ability was determined for the bispecific antibodies E12-TfR1071 obtained in Example 6 against various cell lines with different levels of EGFR expression. Based on the results of Example 9, OE21 cells were used as cells with a high level of EGFR expression, T.Tn cells with an average level of EGFR expression, and U-93 cells with a low level of EGFR expression. As can be seen from Fig. 8A-8C, antibodies E12-TfR1071 showed a high ability to suppress the proliferation of OE21 cells, moderate - in the case of T.Tn cells and did not suppress the proliferation of U-937 cells. From these results it follows that the ability of the E12-TfR1071 antibody to suppress cell proliferation depends on the level of EGFR expression in them.

HepG2 cells were used to determine the inhibition of cell growth by GPC3-TfR bispecific antibodies. As can be seen from Fig. 9, antibodies HN3-TfR1071, HN3-GS-TfR1071 and HN3-GS3-TfR1071 have a very high ability to inhibit cell growth.

To prove the relationship between the ability of GPC3-TfR bispecific antibodies to inhibit cell growth and the level of GPC3 expression, this ability was determined for the bispecific antibodies HN3-TfR1071 obtained in Example 6 against various cell lines with different levels of GPC3 expression; Cell growth inhibition was determined in the same way as in Example 10.

The results obtained with HepG2 cells, in which GPC3 expression is high, are presented in Fig. 10A; The results obtained with HuH-7 cells, in which the expression level of GPC3 is moderate, are presented in Fig. 10B; results obtained with HLE cells in which GPC3 is not expressed are presented in Fig. 10C As can be seen from FIG. 10A-10C, HN3-TfR1071 antibodies significantly suppressed the proliferation of HepG2 cells, in which the level of GPC3 expression is high, showed a moderate ability to suppress the proliferation of HuH-7 cells, in which the level of GPC3 expression is moderate, but did not suppress the proliferation of HLE cells, in which GPC3 is not expressed . From these data it follows that the HN3-TfR1071 antibody exhibits the ability to suppress cell growth depending on the level of GPC3 expression...

It has also been found that for cell surface antigens other than EGFR, bispecific antibodies containing the anti-TfR variable region of the IgG portion exhibit a high cell surface antigen-dependent cell growth inhibitory ability.

Example 13 Inhibition of Transferrin Binding to TfR

antibodies to TfR and bispecific antibodies

to TfR

To establish the mechanism of suppression of cancer cell growth by bispecific antibodies, flow cytometry was used to determine whether EGFR-TfR bispecific antibodies suppress the binding of transferrin to TfR on cancer cells.

OE21 cells were seeded into wells (1 x 10 ⁶ cells) of a 96-well U-bottom plate (manufactured by Falcon, Inc.) with RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS, and the test was added antibody at a concentration of 10 μg/ml and kept the plate in an ice bath for 1 hour. Alexa Fluor 488-labeled mouse transferrin (‎Jackson ImmunoResearch, Inc.) was then added and the plate was left in the ice bath for an additional 1 hour. Then they were washed 2 times with SB solution, the cells were suspended in the same solution and the fluorescence intensity in each well was measured using a flow cytometer, as in Example 9.

In fig. 11(A)-11(D) show the results obtained with cetuximab, TfR1071, E08-TfR1071, E12-TfR1071 and TfR435, respectively. The TfR neutralizing antibody TfR435, which served as a positive control, inhibited transferrin binding to TfR, but the TfR-specific antibody TfR1071 and the EGFR-TfR bispecific antibodies E08-TfR1071 and E12-TfR1071 did not inhibit transferrin binding to TfR as well as cetuximab, which served as a positive control. negative control.

From these data it follows that the EGFR-TfR bispecific antibodies of this invention are not able to neutralize TfR and do not inhibit the binding of transferrin to TfR on the cell surface.

Example 14. Establishment of the mechanism of manifestation of the effectiveness of EGFR-TfR-bispecific antibodies as a drug (EGFR level, TfR level and signaling pathway involving EGFR in cells)

To elucidate the mechanism of inhibition of cancer cell growth by the bispecific antibodies of the present invention, the effect on the EGFR signaling pathway and TfR protein level upon addition of EGFR-TfR bispecific antibodies to cancer cells was determined by Western blotting.

OE21 cells were seeded into wells (1 × 10 ⁶ cells) of a flat-bottomed 6-well plate (manufactured by Falcon, Inc.) with RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS, the antibody to be tested was added, and Cells were cultured at 37°C in an atmosphere containing 5.0% carbon dioxide for 24 hours. After this, the cells were placed in an ice bath, washed once with PBS(-) solution, and then RIPA buffer solution (Thermo Fisher, Inc.) containing Protease/Phosphatase Inhibitor Cocktail (CST Japan, Co., Ltd.) was added; the resulting lysate was collected with a cell scraper.

The collected lysate was centrifuged at 15,000 rpm at 4°C for 5 minutes, the supernatant was collected and a portion of it was determined using the Pierce 660 nm Protein Assay Kit (Thermo Fisher, Inc.). Sample buffer (SDS-PAGE, 6× concentrated and containing a reducing agent (manufactured by Nacalai Tesque, Inc.) was added to the supernatant); the resulting solution, containing 10 μg of protein, was loaded onto precast polyacrylamide gels (ATTO , Inc.) and performed electrophoresis for 25-80 minutes in a PAGE apparatus (manufactured by ATTO, Inc.).

Prestained Protein Size Marker III (Wako Pure Chemical Corporation) was used as a standard for electrophoresis. Gels, filter paper (ATTO, Inc.) and membrane (ATTO, Inc.) were placed in a blotter (ATTO, Inc.) and blotted for 1 hour at 125 mA/gel. After blotting, the membrane was kept in blocking buffer (Pierce, Inc.) on a shaker at room temperature for 1 hour or left overnight at 4°C.

Primary antibodies to various proteins were applied to the membrane (biotinylated anti-Akt kinase antibodies, biotinylated anti-phosphorylated Akt kinase antibodies, biotinylated anti-EGFR antibodies, biotinylated anti-phosphorylated EGFR antibodies, rabbit anti-CD71 antibodies; all from Cell Signaling Trechnology, Inc.) and kept on a shaker at room temperature for 1 hour or left overnight at 4°C.

The membrane was washed 3 times with TBS-T (Wako Pure Chemical Corporation), and secondary antibodies (horseradish peroxidase-streptavidin-conjugated antibodies or horseradish peroxidase-conjugated anti-rabbit IgG; Cell Signaling Trechnology, Inc.) were applied to each first antibodies and kept on a shaker at room temperature for 1 hour. The membrane was then washed 3 times with TBS-T, ECL Prime Western Blotting Detection Regent (GE Healthcare, Inc.) was added, and the membrane was scanned on an Amersham Imager 600 (GE Healthcare, Inc.).

As can be seen from Fig. 12, in the case of antibody E08-TfR1071, the amount of phosphorylated EGFR and phosphorylated AKT kinase was reduced in the same way as in the case of cetuximab, which served as a positive control, while in the case of E12-TfR1071 this reduction was not observed. And the amount of transferrin receptor decreased in the case of E08-TfR1071 and in the case of E12-TfR1071, but did not decrease in the case of cetuximab.

These results demonstrate that E08-TfR1071 is able to inhibit EGFR signaling and induce TfR degradation, while E12-TfR1071 does not inhibit EGFR signaling and only has the ability to induce TfR degradation.

Example 15 Determination of the effect of EGFR-TfR bispecific antibodies

on TfR expression

To determine how bispecific antibodies inhibit the growth of cancer cells, the effect of EGFR-TfR bispecific antibodies on TfR expression on the cell membrane was determined, as described below.

OE21 cells were seeded into wells (1 × 10 ⁶ cells) of a flat-bottomed 6-well plate (manufactured by Falcon, Inc.) with RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS, and the test antibody was added at a concentration of 10 μg/ml and incubated at 37°C for 24 hours. The cells were separated as described in Example 9, transferrin from human serum conjugated to the fluorescent dye Alexa Fluor 488 (Molecular Probes, Inc.) was added to them at a concentration of 5 μg/ml, and the resulting mixture was left for 30 minutes at 4°C C, and then washed as described in Example 9. The amount of transferrin bound to the cell surface was calculated from the fluorescence intensity of single cells measured using a flow fluorimeter.

In fig. 13(A)-13(D) show the results obtained with TfR1071 antibodies, E08-TfR1071 bispecific antibodies and E12-TfR1071 bispecific antibodies, respectively. As can be seen from Fig. 13, the addition of EGFR-TfR bispecific antibodies caused a decrease in the amount of transferrin bound on the cell surface, whereas this was not observed in the case of the TfR1071 antibody.

These results indicate that the inhibition of cancer cell proliferation by the EGFR-TfR bispecific antibodies of the present invention is due to the degradation of TfR, which is reflected in a decrease in the amount of this protein on the surface of these cells.

Example 16. Determination of the effect of EGFR-TfR-bispecific antibodies

on the absorption of iron by cells

To find out what the role of iron is in suppressing the proliferation of cancer cells by EGFR-TfR bispecific antibodies, we did the following.

OE21 cells were seeded into wells (1 × 10 ³ cells) of a flat-bottomed 96-well plate (manufactured by Falcon, Inc.) with RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS, and ferric sulfate was added. and ammonium (Nacalai Tesque, Inc., hereinafter referred to as FAS) to a final concentration of 10 μM along with the test antibody and cultured the cells at 37°C in an atmosphere containing 5.0% carbon dioxide for 4-6 days . ATP levels in viable cells were then determined as described in Example 10.

As can be seen from Fig. 14, if FAS was not added, cell growth was inhibited by cetuximab, TfR435 neutralizing antibodies, and E12-TfR107 bispecific antibodies. If FAS was added, the antiproliferative activity of cetuximab did not change, while the TfR435 neutralizing antibodies and the E12-TfR107 bispecific antibodies did not.

When FAS is added to cells, iron enters the cells without the participation of TfR. Therefore, in the presence of FAS, there is no depletion of the intracellular iron supply that accompanies the blocking of TfR by neutralizing antibodies or the breakdown of TfR under the influence of EGFR-TfR bispecific antibodies, so there is no suppression of cell growth with the participation of TfR. In contrast, the suppression of cell growth caused by inhibition of the signaling pathway involving epidermal growth factor is not affected by the entry of iron into cells due to FAS. Thus, it has been shown that the ability of E12-TfR1071 antibodies to suppress cell growth is not due to inhibition of the signaling pathway involving EGFR, but to the lack of iron entry into the cells - the same as in the case of antibodies that neutralize TfR..

Example 17. Comparison of the effectiveness of EGFR-TfR bispecific antibodies and an inhibitor of the signaling pathway involving EGFR

To compare two mechanisms of cell growth suppression - inhibition of the signaling pathway involving EGFR and inhibition of TfR - experiments were carried out using the IncuCyte intravital cell analysis system (Sartorius AG).

OE21 cells were seeded into wells (1 × 10 ³ cells) of a flat-bottomed 96-well plate with RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS, and the test antibody diluted in the same medium was added to a concentration of 10 µg/ml; cells were incubated at 37°C for 5 days; At the same time, photographs were taken every 2 hours using the IncuCyte system (three fields of view in each well). The medium was replaced, the test antibody was added (concentration 10 μg/ml) and incubated for another 2 days under the same conditions. The medium was replaced again and the cells were incubated for 7 days under the same conditions, but without the test antibody. For each photographing point, the obtained data were analyzed using IncuCyte software.

As can be seen from Fig. 15, in the case of cetuximab, cell growth resumed in medium without the antibody, whereas this was not observed in the case of the bispecific antibody E08-TfR1071 or E12-TfR1071. When the TfR435 antibody, which neutralizes TfR, was added to the cells, the suppression of cell growth persisted in fresh medium without the antibody, although there was a tendency for cell growth to resume; It is suggested that the strong suppression of cell growth that persists in the absence of antibodies in the case of E08-TfR1071 or E12-TfR1071 is due to the fact that these bispecific antibodies cause degradation of TfR.

The results obtained demonstrate that when TfR is inhibited, the suppression of cell growth persists even after the inhibitor disappears, in contrast to the suppression of the signaling pathway involving EGFR.

Example 18. Effect of EGFR-TfR bispecific antibodies

on human bone marrow cells.

To determine the effect of bispecific antibodies E08-TfR1071 and E12-TfR1071 on healthy human bone marrow cells (they do not contain EGFR, but do have TfR), the following was done.

Human bone marrow cells (AllCells, Inc.) were seeded into wells (7500 cells each) of a 96-well plate (manufactured by Falcon, Inc.) containing StemSpan (trade name) SFEM II (STEMCELL Technologies, Inc.), test antibody was added, and Cells were cultured at a temperature of 37°C in an atmosphere containing 5.0% carbon dioxide for 5 days. CellTiter-Glo (registered trade name) Luminescent Cell Vialbility Assay (manufactured by Promega Corporation) was then used and fluorescence intensity was measured using a microplate reader (1420 ARVO Multi-Label Counter, manufactured by WALLAC, Inc.). For each variant of conditions, 2 wells were allocated and the average value was calculated. The fluorescence intensity obtained for a given well reflects the level of ATP in viable cells in that well

To induce differentiation of human bone marrow cells into mature pluripotent hematopoietic stem cells (hereinafter referred to as GEMM), recombinant human stem cell factor (SCF) (PeproTech, Inc.) was added to the cells during culture at a final concentration of 50 ng/ml, human interleukin-3 (IL-3) (Miltenyi Biotec, Inc.) at a final concentration of 10 ng/ml, human interleukin-6 (IL-6) (Sigma-Aldrich Co. LLC) at a final concentration of 20 ng/ml, human colony stimulating granulocyte-macrophage factor (GM-CSF; Miltenyi Biotec, Inc.) at a final concentration of 20 ng/ml, human granulocyte colony-stimulating factor (G-CSF; Miltenyi Biotec, Inc.) at a final concentration of 20 ng/ml, human ligand fms- receptor tyrosine kinase-like 3 (Flt3-LG; Miltenyi Biotec, Inc.) at a final concentration of 50 ng/ml, erythropoietin (Kyowa Hakko Kirin Co., Ltd.) at a final concentration of 3 U/ml, and human thrombopoietin (TPO; Miltenyi Biotec, Inc.) at a final concentration of 30 ng/m.

As can be seen from Fig. 16, the TfR neutralizing antibodies TfR435 inhibit cell growth, while the bispecific antibodies E08-TfR1071 and E12-TfR1071 do not exhibit such activity.

These results confirm that the bispecific antibodies of this invention are not able to inhibit the proliferation of healthy bone marrow cells that express TfRα. Therefore, the bispecific antibodies of this invention should be less toxic to the bone marrow than TfRα neutralizing antibodies.

19. The ability to suppress cell growth in EGFR-TfR-bispecific antibodies with changes in the amino acid sequence

The ability of bispecific antibodies E12-TfR1071, which have changes in the amino acid sequence of VH TfR1071, to inhibit cell growth was determined as described in Example 12, using OE21 cells. First, EGFR-TfR bispecific antibodies with changes in the amino acid sequence were obtained; To do this, we did the following. To obtain an expression vector for bispecific antibodies with changes in the amino acid sequence (with amino acid substitutions), the expression vector, which contained nucleotide sequences encoding the amino acid sequences of the antibody E12-TfR1071 without changes, replaced them with nucleotide sequences encoding the amino acid sequences with substitutions, as described in Example 6. Expression and production of bispecific antibodies with amino acid substitutions was carried out as described in Example 7. The following bispecific antibodies with amino acid substitutions were obtained.

Modified TfR1071 antibodies: Y32A, Y32F, T33A, T33G, L45A, V48A, V50A, I51A, I51L, N52A-A, N52A-D, V55E, D58A, D61P, Q97A, Q97D, P98A, W99A, W99F, W99H, W99Y, Y100A-A, Y100A-F, V102L, P98Y, P98S, P98D, P98Q, P98E, P98T, P98R, P98G, P98K, P98M, P98V, P98L, P98I, P98W, P98F, P98H

Each of the above designations indicates the replacement of an amino acid residue in the modified antibody, namely: [one-letter amino acid designation before the substitution] [position of this amino acid residue in the VH amino acid sequence (SEQ ID NO: 31) of the TfR1071 antibody according to the EU index] [one-letter amino acid designation after replacement]. Hereinafter, amino acid substitutions are referred to in the same manner.

For example, the designation Y32A means that the tyrosine residue (denoted by the letter Y) at position 32 (according to the EU index) is replaced by an alanine residue (denoted by the letter A). The designations 52A and 100A mean that this amino acid residue is located in positions “52-53” and “100-101”, respectively (according to the EU index). For example, the designation N52A-D means that at position 52A the EU suffix residue is changed from N to A, and the designation Y100A-A means that at position 100A the EU suffix is changed from Y to A.

Cancer cells were seeded into wells (1 × 10 ³ cells) of a flat-bottomed 96-well plate (manufactured by Falcon, Inc.), and the test antibody diluted to various concentrations with RPMI 1640 medium (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS, and cells were cultured at 37°C in an atmosphere containing 5.0% carbon dioxide for 4-6 days. Cell Counting Kit-8 reagent (Dojindo, Inc.) was then added and absorbance was measured using a microplate reader (1420 ARVO Multi-Label Counter, manufactured by WALLAC, Inc.). For each variant of conditions, 3 wells were allocated and the average value was calculated. The fluorescence intensity obtained for a given well reflects the number of viable cells in that well. Anti-DNP antibodies were used as a negative control. The results of the influence of the above bispecific antibodies on cell growth are presented in Fig. 17A and 17B.

As can be seen from Fig. 17A and 17B, modified antibodies of clone TfR1071 with amino acid substitutions N52A-A and N52A-D did not exhibit the ability to suppress cell growth, modified antibodies with amino acid substitutions V55E, W99A and W99H had this ability to a small extent, and all other modified antibodies had a high the ability to inhibit cell growth compared to that before the amino acid sequence was changed.

These results demonstrate that in the E12-TfR1071 bispecific antibodies of the present invention, N52 is important for the ability to inhibit cell growth. Further, even if the P98 residue is replaced by any other amino acid residue, an equivalent ability of the antibody to inhibit cell growth is observed; Presumably, this amino acid residue can be replaced by any other naturally occurring amino acid residue.

Example 20 Epitope Identification

recognized by anti-TfR antibody

The epitope recognized by the anti-TfR 1071 antibody was identified as follows.

Using the method described in Example 7, based on the sequence information given in WO 2014/189973, antibodies to TfR 15G11v5, 7A4v15 and 16F6v4, which bind to the apical domain of human TfR, and 7G7v1, which recognizes its protease-like domain, were obtained

The extracellular domain of human TfR was obtained, described in Example 1. Next, a chimeric protein was obtained in which the apical or protease-like domain of the extracellular domain of human TfR was replaced with the corresponding domain of mouse TfR (containing the amino acid sequence presented in the GenBank database under the accession number NP_001344227, or amino acid sequence shown by SEQ ID NO: 87). Namely, the nucleotide sequence encoding this amino acid sequence was subcloned into the pCI-OtCM vector, which included a histidine tag; thus obtaining an expression vector for the desired protein. Using this vector, in this way. described in Example 1, the proteins His-huTfR, huTfR with a mouse apical domain (SEQ ID NO: 88) and huTfR with a mouse protease-like domain (SEQ ID NO: 89) were obtained, each of which was added with a histidine tag.

The designation His-huTfR represents the extracellular domain of human TfR with a histidine tag; the name “mouse apical domain huTfR” represents the extracellular domain of a human TfR with a histidine tag in which the apical domain has been replaced by the corresponding mouse sequence; the name “mouse protease-like domain huTfR” represents the extracellular domain of a human TfR with a histidine tag in which the protease-like domain has been replaced by the corresponding mouse sequence.

Similarly, chimeric TfR A01-A16 were obtained, in which some amino acid residues in the apical domain of human TfR were replaced by the corresponding amino acid residues characteristic of mouse TfR. The resulting chimeric proteins are presented in Table 5; their amino acid sequences are represented by SEQ ID NO: 90-105, respectively.

Table 5 lists the amino acid substitutions made for each chimeric protein. For example, in the case of the A01 protein, the entry in the right column of the table Q285K-T286N-T362S means that in the amino acid sequence of human TfR represented by SEQ ID NO: 6, at position 285 the amino acid residue Q is replaced by K, at position 286 the amino acid residue T) is replaced to N and at position 362 the amino acid residue T is replaced by S. In cases where there is no corresponding residue in the mouse TfR, V210 of A08, for example V210 or A08, the amino acid was removed.

Table 5. Chimeric TfR proteins for epitope identification

A01 Q285K-T286N-T362S A02 D352S-S355A-D356R-K358N A03 D245E-K261E A04 D245E-K261E-D356R A05 P249S-L274F A06 E272Q A07 M365L-V366E-E369Q A08 N348K-K371Q-V210-Y211D-D204Q A09 A225P-A226T-T227E-T229S A10 A225P-A226T-T227E A11 D194S-A196IG-S378K A12 G217E-S296A A13 D204Q-K205S A14 D204Q-K205S-E369Q A15 S199M-I201T-L212P-N215S A16 S199M-T376I-T227E

Epitope analysis was then performed for various TfR antibodies. First, the reactivity of anti-TfR antibodies produced by human and murine TfR/CHO cells obtained as described in Example 2 was determined by flow cytometry as described in Example 9. Antibodies R327, TfR1071, cyno186, cyno292 and 2230 were found to bind to human TfR, but do not bind to mouse. In addition, it is known from WO 2014/189973 that antibodies 15G11v5, 7A4v15, 16F6v4 and 7G7v1 do not cross-react with the mouse protein.

The binding activity of anti-TfR antibodies to various chimeric TfR proteins prepared as described above was then determined by ELISA; thus, the binding domain of anti-TfR antibodies and the epitope they recognize were identified. If no antibody binding was observed with the chimeric TfR protein, the amino acid residue that was replaced in this chimeric protein was considered to be part of the epitope.

ELISA was performed as follows. Antigenic protein was immobilized on a 96-well immunoassay plate (NUNC, Inc.), nonspecific binding was blocked with 1% (w/v) pH 7.0 KCl-free BSA-PBS(-) (hereinafter referred to as blocking solution; Nacalai Tesque, Inc.) at room temperature, after which anti-TfR antibody diluted in the same solution was added and the reaction was left for 1 hour at room temperature. Then washed with 0.05% (w/v) -tPBS (1x) without KCl (pH 7.2) (hereinafter referred to as wash solution; Nacalai Tesque, Inc.), anti-human IgG was added ( Fc) goat IgG Fab”-HRP (IBL, Inc). diluted in blocking solution and left for 1 hour at room temperature for the reaction to occur. Wash with wash solution, add 1-Step (registered trade name) Ultra TMB-ELISA Substrate Solution (Thermo, Inc.) and leave at room temperature to allow color to develop. The color reaction was stopped by adding hydrochloric acid solution (5 N) and absorbance was measured at 450 nm (reference wavelength 570 nm) using a plate reader (EPOCH 2; BioTek Instruments, Inc.).

The results obtained are presented in Table 6 (the designation N/A means that there is no data). The difference in absorption values at a wavelength of 450 nm and at a reference wavelength of 570 nm was taken as a measure of binding activity to the antigenic protein; those cases where the value obtained by dividing the value of binding activity with a particular chimeric TfR by the value of binding activity with His-huTfR was 0.5 or more are indicated (++); those cases where the specified value was 0.2 or more, but less than 0.5, are indicated (+); those cases where this value was less than 0.2 are indicated (-).

Table 6. Determination of epitopes for various antibodies to TfR

to TfR

Antibody clone Linking with
antigenic protein
according to ELISA Binding domain Linking with
chimeric protein TfR
according to ELISA Binding site
in the apical
domain huTfR
with mouse
apical domain huTfR
with mouse
protease
similar domain A02 A03 A04 A05 A07 A14 T14 - ++ Apical ++ ++ + - ++ ++ A05 R327 - ++ Apical - ++ + ++ - ++ A02, A07 TfR1007 - ++ Apical - ++ + ++ - ++ A02, A07 TfR1071 - ++ Apical - ++ + ++ - ++ A02, A07 cyno186 - ++ Apical - ++ + ++ - - A02, A07, A14 cyno292 - ++ Apical - ++ ++ ++ - ++ A02, A07 2230 ++ - Protease-like ++ ++ ++ ++ ++ ++ (Protease-like) 15G11v5 - ++ Apical - - - ++ + ++ A02, A03, A04 7A4v15 - ++ Apical - ++ + ++ + ++ A02 16F6v4 - ++ Apical - ++ + ++ ++ ++ A02 7G7v1 ++ - Proteazo-like N/A N/A N/A N/A N/A N/A (Protease-like)

These results demonstrate that antibodies T14, R327, TfR1071, cyno186, cyno292, 15G11v5, 7A4v15, and 16F6v4 recognize the apical domain of TfR, and antibodies 2230 and 7G7v1 recognize the protease-like domain. Among the antibodies that recognize the apical domain, antibody T14 recognizes amino acid substitution sites in A05; R327, TfR1007, TfR1071, cyno292 - A02 and A07; cyno186 - in A02, A07 and A14; 15G11v5 – in A02, A03 and A04; 7A4v15 and 16F6v4 - in A02.

It was shown that the amino acid residues recognized by the TfR1071 antibody are those that are replaced in the chimeric proteins A02 and A07. Thus, it is believed that the TfR1071 antibody recognizes seven amino acid residues, namely D352, S355, D356, K358, M365, V366 and E369. The same can be said about antibodies R327, TfR1007 and cyno292.

Example 21. Epitope recognized by anti-TfR antibody and the ability of EGFR-TfR bispecific antibodies to inhibit cell growth

To study the relationship between the ability of the bispecific antibodies of this invention to inhibit cell growth and the paratope of the variable region of the portion of IgG that binds to the TfR epitope, bispecific antibodies E12-TfR were prepared using different anti-TfR antibodies recognizing different epitopes and their ability was determined suppress cell growth.

The light chain of the anti-TfR antibodies 15G11v5, 7A4v15, 16F6v4 and 7G7v1 and the light chain of the anti-EGFR E12 antibody are different, so bispecific antibodies having the structure shown in FIG. 18(A), for which we applied the method described in Example 6. To determine the binding activity of bispecific antibodies, we proceeded as described in Example 8, namely, antibodies were immobilized on the CM5 sensor chip to detect proteins with a histidine tag Penta-His Antibody (QIAGEN, Inc.), the His-huTfR protein was added and the binding activity of the analyte, a bispecific antibody, was determined; Association/dissociation constants and equilibrium constants were calculated for each of the bispecific antibodies. The results are presented in Table 7.

Table 7. Binding activity of bispecific antibodies to TfR

k _a k _d K _D E12-TfR1071 6.02E+05 9.73E-03 1.62E-08 E12-15G11v5 1.68E+05 2.03E-04 1.21E-09 E12-7A4v15 1.04E+05 8.75E-04 8.39E-09 E12-16F6v4 2.86E+04 7.88E-04 2.76E-08 E12-7G7v1 1.23E+05 1.07E-03 8.74E-09 Cetuximab-TfR1071 5.48E+05 1.05E-02 1.92E-08 Panitumumab-TfR1071 4.84E+05 1.00E-02 2.07E-08 Necitumumab-TfR1071 4.77E+05 9.19E-03 1.93E-08 Nimotuzumab-TfR1071 5.17E+05 9.52E-03 1.84E-08

As can be seen from Table 7, all bispecific antibodies obtained strongly bound to TfR.

The results of determining the inhibition of OE21 cell proliferation by the obtained bispecific antibodies, which was carried out as described in Example 19, are depicted in FIG. 19A and 19B.

As can be seen from Fig. 19A and 19B, the maximum activity of EGFR-TfR bispecific antibodies based on antibodies R327, TfR1007, TfR1071, cyno186 and cyno292 was higher than that of the other bispecific antibodies. Judging by these results and the results of Example 20, bispecific antibodies based on antibodies R327, TfR1007, TfR1071 and cyno292, which recognize epitopes in the structure of which involve sites of amino acid substitutions in proteins A02 and A07, as well as cyno186, which recognizes, have a high ability to suppress cell growth. epitopes, the structure of which involves sites of amino acid substitutions in proteins A02, A07 and A14. Thus. It has been shown that for a high ability to inhibit cell growth, the antibody must recognize an epitope formed with the participation of amino acid residues replaced in proteins A02 and A07.

In addition, as can be seen from FIG. 19A, among those bispecific antibodies that recognize an epitope formed with the participation of amino acid residues. replaced in proteins A02 and A07, bispecific antibodies based on antibodies TfR1071 and R327 had the greatest activity starting from the lowest concentration. In other words, clones TfR1071 and R327 turned out to be the best.

Example 22. Inhibition of cell growth by EGFR-TfR bispecific antibodies based on various antibodies to EGFR and the TfR1071 antibody

To determine whether the TfR1071 component is essential for the ability of bispecific antibodies to inhibit cell growth, EGFR-TfR bispecific antibodies were generated using commercially available anti-EGFR antibodies capable of inhibiting this receptor and the anti-TfR antibody TfR1071 and determined their ability suppress cell growth.

Bispecific antibodies were obtained from antibodies to EGFR (cetuximab, panitumumab, necitumumab and nimotuzumab) and antibodies to TfR TfR1071, as described in Example 6. The sequences of the variable regions of cetuximab, panitumumab, necitumumab and nimotuzumab were used, described in publications WO 1996/040210, WO 1998/050433, WO 2011/116387 and US Patent Application No. 2012/0308576, respectively. The structure of these antibodies corresponds to that shown in Fig. 1(C).

The results of determining the inhibition of cell growth by the obtained bispecific antibodies, as measured by the inhibition of cancer cell proliferation according to Example 20, are shown in FIG. 20.

As can be seen from Fig. 20, EGFR-TfR bispecific antibodies had a high ability to inhibit cell growth, in which the CDR of the TfR1071 antibody was present, and the variable region could be from any antibody to EGFR.

Based on these results, it can be assumed that in order for a bispecific antibody to have a high ability to inhibit cell growth, its IgG part must contain the CDR of the TfR1071 antibody.

Example 23 Comparison of the ability of heterodimeric bispecific antibodies to inhibit cell growth

To determine the importance of the binding valence of the bispecific antibodies of the present invention to TfR and to EGFR for their ability to inhibit cell growth, a heterodimeric bispecific antibody was prepared that had one antigen-binding domain of the E12 anti-EGFR antibody and one antigen-binding domain of the antibody TfR1071 anti-TfR (hereinafter referred to as heterodimeric antibody) and its ability to inhibit cell growth was determined. In fig. 18(B) is a schematic diagram of the structure of this heterodimeric antibody.

The heterodimeric bispecific antibody Hetero E12-TfR107, which had one antigen-binding domain of the E12 anti-EGFR antibody and one antigen-binding domain of the anti-TfR antibody TfR107, was prepared by the method described in the patent literature (see US patent application No. 2014/0348839) ; its ability to inhibit cell growth was determined as described in Example 11. The results are presented in FIG. 21.

As can be seen from Fig. 21, the maximum activity and specific cell growth inhibition activity of Hetero E12-TfR1071 are weaker than those of the bispecific antibody E12-TfR1071. The results obtained demonstrate that the bispecific antibodies of this invention, which have a valence of 2 for each of the antigens - TfR and EGFR, have a higher ability to inhibit cell growth than heterodimeric antibodies, which have a valence of 2 for each of the antigens. antigens – to TfR and to EGFR – is equal to 1.

Example 24 Binding Activity

bispecific antibody E12-TfR1071YE with TfR

Bispecific antibody E12-TfR1071YE having a VH sequence (SEQ ID NO: 106) in which the amino acid at position 1 of the VH sequence (SEQ ID NO: 31) of TfR1071 is replaced from tyrosine (Y) to (E (glutamic acid). The binding activity of this bispecific antibody to human TfR by ELISA as described in Example 20. The binding activity of the bispecific antibody E12-TfR1071YE was found to be comparable to that of E12-TfR1071.

The invention has been described herein in detail in its specific aspects, but one skilled in the art will appreciate that various changes and modifications are possible without departing from the spirit and scope of the invention. This application is based on a Japanese patent application filed on December 28, 2018 (Patent Application No. 2018-248334), which is incorporated herein by reference in its entirety.

List of sequences

SEQ ID NO: 1: Nucleotide sequence of the extracellular domain of human TfR

SEQ ID NO: 2: amino acid sequence of the extracellular domain of human TfR

SEQ ID NO: 3: Nucleotide sequence of the extracellular domain of simian TfR

SEQ ID NO: 4: Amino acid sequence of the extracellular domain of simian TfR

SEQ ID NO: 5: Nucleotide sequence of human TfR

SEQ ID NO: 6: Amino acid sequence of human TfR

SEQ ID NO: 7: Cynomolgus TfR Nucleotide Sequence

SEQ ID NO: 8: Cynomolgus TfR amino acid sequence

SEQ ID NO: 9: Nucleotide sequence of the extracellular domain of human EGFR (including signal sequence)

SEQ ID NO: 10: amino acid sequence of the extracellular domain of human EGFR (including signal sequence)

SEQ ID NO: 11: nucleotide sequence of simian EGFR extracellular domain (including signal sequence)

SEQ ID NO: 12: amino acid sequence of the extracellular domain of simian EGFR (including signal sequence)

SEQ ID NO: 13: Nucleotide sequence of human EGFR

SEQ ID NO: 14: amino acid sequence of human EGFR

SEQ ID NO: 15: VL A27 nucleotide sequence

SEQ ID NO: 16: amino acid sequence of VL A27

SEQ ID NO: 17: amino acid sequence of LCDR1 A27

SEQ ID NO: 18: amino acid sequence of LCDR2 A27

SEQ ID NO: 19: amino acid sequence of LCDR3 A27

SEQ ID NO: 20: Nucleotide sequence of VH PSM4072

SEQ ID NO: 21: amino acid sequence of VH PSM4072

SEQ ID NO: 22: amino acid sequence of HCDR1 PSM4072

SEQ ID NO: 23: amino acid sequence of HCDR2 PSM4072

SEQ ID NO: 24: amino acid sequence of HCDR3 PSM4072

SEQ ID NO: 25: Nucleotide sequence of VH R327

SEQ ID NO: 26: amino acid sequence of VHR327

SEQ ID NO: 27: amino acid HCDR1 R327

SEQ ID NO: 28: amino acid sequence of HCDR2 R327

SEQ ID NO: 29: amino acid sequence of HCDR3 R327

SEQ ID NO: 30: Nucleotide sequence of VH TfR1071

SEQ ID NO: 31: amino acid sequence of VH TfR1071

SEQ ID NO: 32: amino acid sequence of HCDR1 TfR1071

SEQ ID NO: 33: amino acid sequence of HCDR2 TfR1071

SEQ ID NO: 34: amino acid sequence of HCDR3 TfR1071

SEQ ID NO: 35: Nucleotide sequence of VH TfR4016

SEQ ID NO: 36: amino acid sequence of VH TfR4016

SEQ ID NO: 37: amino acid sequence of HCDR1 TfR4016

SEQ ID NO: 38: amino acid sequence of HCDR2 TfR4016

SEQ ID NO: 39: amino acid sequence of HCDR3 TfR4016

SEQ ID NO: 40: Nucleotide sequence of VH cyno186

SEQ ID NO: 41: amino acid sequence of VH cyno186

SEQ ID NO: 42: amino acid sequence of HCDR1 cyno186

SEQ ID NO: 43: amino acid sequence of HCDR2 cyno186

SEQ ID NO: 44: amino acid sequence of HCDR3 cyno186

SEQ ID NO: 45: Nucleotide sequence of VH cyno292

SEQ ID NO: 46: amino acid sequence of VH cyno292

SEQ ID NO: 47: amino acid sequence of HCDR1 cyno292

SEQ ID NO: 48: amino acid sequence of HCDR2 cyno292

SEQ ID NO: 49: amino acid sequence of HCDR3 cyno292

SEQ ID NO: 50: amino acid sequence of VH TfR1007

SEQ ID NO: 51: amino acid sequence of VH cyno163

SEQ ID NO: 52: amino acid sequence of VH 2230

SEQ ID NO: 53: amino acid sequence of VL2230

SEQ ID NO: 54: amino acid sequence of VH T14

SEQ ID NO: 55: amino acid sequence of VH TfR434

SEQ ID NO: 56: amino acid sequence of VH TfR435

SEQ ID NO: 57: amino acid sequence of VL TfR434 and TfR435

SEQ ID NO: 58: VH E08 nucleotide sequence

SEQ ID NO: 59: amino acid sequence of VH E08

SEQ ID NO: 60: amino acid sequence of HCDR1 E08

SEQ ID NO: 61: amino acid sequence of HCDR2 E08

SEQ ID NO: 62: amino acid sequence of HCDR3 E08

SEQ ID NO: 63: VH E12 nucleotide sequence

SEQ ID NO: 64: amino acid sequence of VH E12

SEQ ID NO: 65: amino acid sequence of HCDR1 E12

SEQ ID NO: 66: HCDR2 E12 amino acid sequence

SEQ ID NO: 67: HCDR3 E12 amino acid sequence

SEQ ID NO: 68: VH E17 nucleotide sequence

SEQ ID NO: 69: amino acid sequence of VH E17

SEQ ID NO: 70: amino acid sequence of HCDR1 E17

SEQ ID NO: 71: HCDR2 E17 amino acid sequence

SEQ ID NO: 72: amino acid sequence of HCDR3 E17

SEQ ID NO: 73: amino acid sequence of VH KME07

SEQ ID NO: 74: amino acid sequence of VH KME09

SEQ ID NO: 75: amino acid sequence of VH KME11

SEQ ID NO: 76: VH amino acid sequence of cetuximab

SEQ ID NO: 77: amino acid sequence of VL cetuximab

SEQ ID NO: 78: amino acid sequence of VH HN3

SEQ ID NO: 79: Linker amino acid sequence

SEQ ID NO: 80: Linker amino acid sequence

SEQ ID NO: 81: Linker amino acid sequence

SEQ ID NO: 82: Linker amino acid sequence

SEQ ID NO: 83: Nucleotide sequence of IgG4PE R409K constant region

SEQ ID NO: 84: amino acid sequence of IgG4PE R409K constant region

SEQ ID NO: 85: nucleotide sequence with modified codons of the CH1 region of IgG4

SEQ ID NO: 86: Amino acid sequence of IgG4PE constant region

SEQ ID NO: 87: amino acid sequence of mouse TfR

SEQ ID NO: 88: amino acid sequence of a chimeric protein comprising the extracellular domain of human TfR and the apical domain of mouse TfR

SEQ ID NO: 89: amino acid sequence of a chimeric protein comprising the extracellular domain of human TfR and the protease-like domain of mouse TfR

SEQ ID NO: 90: amino acid sequence A01

SEQ ID NO: 91: amino acid sequence A02

SEQ ID NO: 92: amino acid sequence A03

SEQ ID NO: 93: amino acid sequence A04

SEQ ID NO: 94: amino acid sequence A05

SEQ ID NO: 95: amino acid sequence A06

SEQ ID NO: 96: amino acid sequence A07

SEQ ID NO: 97: amino acid sequence A08

SEQ ID NO: 98: Amino acid sequence of A09

SEQ ID NO: 99: A10 amino acid sequence

SEQ ID NO: 100: amino acid sequence A11

SEQ ID NO: 101: A12 amino acid sequence

SEQ ID NO: 102: amino acid sequence of A13

SEQ ID NO: 103: A14 amino acid sequence

SEQ ID NO: 104: A15 amino acid sequence

SEQ ID NO: 105: A16 amino acid sequence

SEQ ID NO: 106: amino acid sequence of VH TfR1071 starting with EVQL

SEQ ID NO: 107: amino acid sequence of HCDR1 TfR1007

SEQ ID NO: 108: amino acid sequence of HCDR2 TfR1007

SEQ ID NO: 109: amino acid sequence of HCDR3 TfR1007

SEQ ID NO: 110: VH amino acid sequence of cetuximab

SEQ ID NO: 111: amino acid sequence of VL cetuximab

SEQ ID NO: 112: VH amino acid sequence of panitumumab

SEQ ID NO: 113: amino acid sequence of VL panitumumabaa

SEQ ID NO: 114: VH amino acid sequence of necitumumab

SEQ ID NO: 115: amino acid sequence of VL necitumumab

SEQ ID NO: 116: VH amino acid sequence of nimotuzumab

SEQ ID NO: 117: Amino acid sequence of Nimotuzumab VL

--->

LIST OF SEQUENCES

<110> Kyowa Kirin Co., Ltd.

<120> Bispecific antibody binding to TfR

<130> W528470

<150> JP2018-248334

<151> 2018-12-28

<160> 117

<170> Patent In version 3.5

<210> 1

<211> 1643

<212> DNA

<213> Homo sapiens

<400> 1

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120

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300

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360

ctgtccagac aatctccaga gctgctgcag aaaagctgtt tgggaatatg gaaggagact

420

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480

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540

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600

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660

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720

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780

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840

acttcaaggt ttctgccagc ccactgttgt atacgcttat tgagaaaaca atgcaaaatg

900

tgaagcatcc ggttactggg caatttctat atcaggacag caactgggcc agcaaagttg

960

agaaactcac tttagacaat gctgctttcc ctttccttgc atattctgga atcccagcag

1020

tttctttctg tttttgcgag gacacagatt atccttattt gggaaccacc atggacacct

1080

ataaggaact gattgagagg attcctgagt tgaacaaagt ggcacgagca gctgcagagg

1140

tcgctggtca gttcgtgatt aaactaaccc atgatgttga attgaacctg gactatgaga

1200

ggtacaacag ccaactgctt tcatttgtga gggatctgaa ccaatacaga gcagacataa

1260

aggaaatggg cctgagttta cagtggctgt attctgctcg tggagacttc ttccgtgcta

1320

cttccagact aacaacagat ttcgggaatg ctgagaaaac agacagattt gtcatgaaga

1380

aactcaatga tcgtgtcatg agagtggagt atcacttcct ctctccctac gtatctccaa

1440

aagagtctcc tttccgacat gtcttctggg gctccggctc tcacacgctg ccagctttac

1500

tggagaactt gaaactgcgt aaacaaaata acggtgcttt taatgaaacg ctgttcagaa

1560

accagttggc tctagctact tggactattc agggagctgc aaatgccctc tctggtgacg

1620

tttgggacat tgacaatgag ttt

1643

<210> 2

<211> 672

<212>PRT

<213> Homo sapiens

<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

<210> 3

<211> 2016

<212> DNA

<213> Monkey

<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 atggctagag

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

<210> 4

<211> 672

<212>PRT

<213> Monkey

<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

<210> 5

<211> 2280

<212> DNA

<213> Homo sapiens

<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

<210> 6

<211> 760

<212>PRT

<213> Homo sapiens

<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 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

<210> 7

<211> 2280

<212> DNA

<213> Cynomolgus monkey

<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 cccagagat 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

tttttgaggg 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

<210> 8

<211> 760

<212>PRT

<213> Cynomolgus monkey

<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

<210> 9

<211> 1914

<212> DNA

<213> Homo sapiens

<400> 9

atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg

60

gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag

120

ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg

180

gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag

240

accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct

300

ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca

360

gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta

420

caggaaatcc tgcatggcgc cgtgcggttc agcaacaacc ctgccctgtg caacgtggag

480

agcatccagt ggcgggacat agtcagcagt gactttctca gcaacatgtc gatggacttc

540

cagaaccacc tgggcagctg ccaaaagtgt gatccaagct gtcccaatgg gagctgctgg

600

ggtgcaggag aggagaactg ccagaaactg accaaaatca tctgtgccca gcagtgctcc

660

gggcgctgcc gtggcaagtc ccccagtgac tgctgccaca accagtgtgc tgcaggctgc

720

acaggccccc ggggagagcga ctgcctggtc tgccgcaaat tccgagacga agccacgtgc

780

aaggacacct gccccccact catgctctac aaccccacca cgtaccagat ggatgtgaac

840

cccgaggca aatacagctt tggtgccacc tgcgtgaaga agtgtccccg taattatgtg

900

gtgacagatc acggctcgtg cgtccgagcc tgtggggccg acagctatga gatggaggaa

960

gacggcgtcc gcaagtgtaa gaagtgcgaa gggccttgcc gcaaagtgtg taacggaata

1020

ggtattggtg aatttaaaga ctcactctcc ataaatgcta cgaatattaa acacttcaaa

1080

aactgcacct ccatcagtgg cgatctccac atcctgccgg tggcatttag gggtgactcc

1140

ttcacacata ctcctcctct ggatccacag gaactggata ttctgaaaac cgtaaaggaa

1200

atcacaggt ttttgctgat tcaggcttgg cctgaaaaca ggacggacct ccatgccttt

1260

gagaacctag aaatcatacg cggcaggacc aagcaacatg gtcagttttc tcttgcagtc

1320

gtcagcctga acataacatc cttgggatta cgctccctca aggagataag tgatggagat

1380

gtgataattt caggaaacaa aaatttgtgc tatgcaaata caataaactg gaaaaaactg

1440

tttgggacct ccggtcagaa aaccaaaatt ataagcaaca gaggtgaaaa cagctgcaag

1500

gccacaggcc aggtctgcca tgccttgtgc tcccccgagg gctgctgggg cccggagccc

1560

agggactgcg tctcttgccg gaatgtcagc cgaggcaggg aatgcgtgga caagtgcaac

1620

cttctggagg gtgagccaag ggagtttgtg gagaactctg agtgcataca gtgccaccca

1680

gagtgcctgc ctcaggccat gaacatcacc tgcacaggac ggggaccaga caactgtatc

1740

cagtgtgccc actacattga cggcccccac tgcgtcaaga cctgcccggc aggagtcatg

1800

ggagaaaaca acaccctggt ctggaagtac gcagacgccg gccatgtgtg ccacctgtgc

1860

catccaaact gcacctacgg atgcactggg ccaggtcttg aaggctgtcc aacg

1914

<210> 10

<211> 638

<212>PRT

<213> Homo sapiens

<400> 10

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

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

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

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

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

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

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

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

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

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

370 375 380

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

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

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

515 520 525

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

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr

625 630 635

<210> 11

<211> 1914

<212> DNA

<213> Monkey

<400> 11

atgcgaccct ccgggacggc cggggccgcg ctcctggcgc tgctggctgc gctctgcccc

60

gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa actcacgcag

120

ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg

180

gtccttggga atttggaaat tacctacgtg cagaggaatt atgatctttc cttcttaaag

240

accatccagg aggtggctgg ttatgtcctc atcgccctca acacagtgga gcggattcct

300

ttggaaaacc tgcagatcat cagaggaaac atgtactatg aaaattccta tgccttagca

360

gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaactta

420

caggaaatcc tgcatggcgc cgtgcggttc agcaacaacc ctgccctgtg caacgtggag

480

agcatccagt ggcgggacat agtcagcagc gagtttctca gcaacatgtc gatggacttc

540

cagaaccacc tgggcagctg ccaaaagtgt gatccaagct gtcccaatgg gagctgctgg

600

ggtgcaggag aggagaactg ccagaaactg accaaaatca tctgtgccca gcagtgctcc

660

gggcgctgcc gcggcaagtc ccccagtgac tgctgccaca accagtgtgc cgcgggctgc

720

acgggccccc ggggagagcga ctgcctggtc tgccgcaaat tccgagacga agccacgtgc

780

aaggacacct gccccccact catgctctac aaccccacca cataccagat ggatgtgaac

840

cccgaggca aatacagctt tggtgccacc tgcgtgaaga agtgtccccg taattatgtg

900

gtgacagatc acggctcgtg cgtccgagcc tgcggggccg acagctatga gatggaggaa

960

gacggcgtcc gcaagtgtaa gaagtgcgaa gggccttgcc gcaaagtgtg taatggaata

1020

ggtattggtg aatttaaaga cacactctcc ataaatgcta caaatattaa acacttcaaa

1080

aactgcacct ccatcagtgg cgatctccac atcctgccgg tggcatttag gggtgactcc

1140

ttcacacaca ctccgcctct ggatccacag gaactggata ttctgaaaac cgtaaaggaa

1200

atcacaggt ttttgctgat tcaggcttgg cctgaaaaca ggacggacct ccatgctttt

1260

gagaacctag aaatcatacg tggcaggacc aagcaacacg gtcagttttc tcttgcggtc

1320

gtcagcctga acataacatc cttgggatta cgctccctca aggagataag cgatggagat

1380

gtgataattt caggaaacaa aaatttgtgc tatgcaaata caataaactg gaaaaaactg

1440

tttgggacct ccagtcagaa aaccaaaatt ataagcaaca gaggtgaaaa cagctgcaag

1500

gccacgggcc aggtctgcca tgccttgtgc tcccccgagg gctgctgggg cccggagccc

1560

agggactgcg tctcctgtca gaatgtcagc cgaggcagag aatgcgtgga caagtgcaac

1620

atcctggagg gcgagccaag ggagtttgtg gagaactctg agtgcataca gtgccaccca

1680

gaatgcctgc cccaggtcat gaacatcacc tgcacaggac ggggaccaga caactgtatc

1740

cagtgtgccc actacattga cggcccccac tgcgtcaaga cctgcccagc aggagtcatg

1800

ggagaaaaca acaccctggt ctggaagtac gcagacgccg gccacgtgtg ccacttgtgc

1860

catccaaact gcacctacgg atgcactggg ccaggtcttg aaggctgtgc aagg

1914

<210> 12

<211> 638

<212>PRT

<213> Monkey

<400> 12

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

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

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

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

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Glu Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

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

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

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

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

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

370 375 380

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

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Ser Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

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

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Ile Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Val Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Ala Arg

625 630 635

<210> 13

<211> 3630

<212> DNA

<213> Homo sapiens

<400> 13

atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg

60

gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag

120

ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg

180

gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag

240

accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct

300

ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca

360

gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta

420

caggaaatcc tgcatggcgc cgtgcggttc agcaacaacc ctgccctgtg caacgtggag

480

agcatccagt ggcgggacat agtcagcagt gactttctca gcaacatgtc gatggacttc

540

cagaaccacc tgggcagctg ccaaaagtgt gatccaagct gtcccaatgg gagctgctgg

600

ggtgcaggag aggagaactg ccagaaactg accaaaatca tctgtgccca gcagtgctcc

660

gggcgctgcc gtggcaagtc ccccagtgac tgctgccaca accagtgtgc tgcaggctgc

720

acaggccccc ggggagagcga ctgcctggtc tgccgcaaat tccgagacga agccacgtgc

780

aaggacacct gccccccact catgctctac aaccccacca cgtaccagat ggatgtgaac

840

cccgaggca aatacagctt tggtgccacc tgcgtgaaga agtgtccccg taattatgtg

900

gtgacagatc acggctcgtg cgtccgagcc tgtggggccg acagctatga gatggaggaa

960

gacggcgtcc gcaagtgtaa gaagtgcgaa gggccttgcc gcaaagtgtg taacggaata

1020

ggtattggtg aatttaaaga ctcactctcc ataaatgcta cgaatattaa acacttcaaa

1080

aactgcacct ccatcagtgg cgatctccac atcctgccgg tggcatttag gggtgactcc

1140

ttcacacata ctcctcctct ggatccacag gaactggata ttctgaaaac cgtaaaggaa

1200

atcacaggt ttttgctgat tcaggcttgg cctgaaaaca ggacggacct ccatgccttt

1260

gagaacctag aaatcatacg cggcaggacc aagcaacatg gtcagttttc tcttgcagtc

1320

gtcagcctga acataacatc cttgggatta cgctccctca aggagataag tgatggagat

1380

gtgataattt caggaaacaa aaatttgtgc tatgcaaata caataaactg gaaaaaactg

1440

tttgggacct ccggtcagaa aaccaaaatt ataagcaaca gaggtgaaaa cagctgcaag

1500

gccacaggcc aggtctgcca tgccttgtgc tcccccgagg gctgctgggg cccggagccc

1560

agggactgcg tctcttgccg gaatgtcagc cgaggcaggg aatgcgtgga caagtgcaac

1620

cttctggagg gtgagccaag ggagtttgtg gagaactctg agtgcataca gtgccaccca

1680

gagtgcctgc ctcaggccat gaacatcacc tgcacaggac ggggaccaga caactgtatc

1740

cagtgtgccc actacattga cggcccccac tgcgtcaaga cctgcccggc aggagtcatg

1800

ggagaaaaca acaccctggt ctggaagtac gcagacgccg gccatgtgtg ccacctgtgc

1860

catccaaact gcacctacgg atgcactggg ccaggtcttg aaggctgtcc aacgaatggg

1920

cctaagatcc cgtccatcgc cactgggatg gtggggggccc tcctcttgct gctggtggtg

1980

gccctgggga tcggcctctt catgcgaagg cgccacatcg ttcggaagcg cacgctgcgg

2040

aggctgctgc aggagaggga gcttgtggag cctcttacac ccagtggaga agctcccaac

2100

caagctctct tgaggatctt gaaggaaact gaattcaaaa agatcaaagt gctgggctcc

2160

ggtgcgttcg gcacggtgta taagggactc tggatcccag aaggtgagaa agttaaaatt

2220

cccgtcgcta tcaaggaatt aagagaagca acatctccga aagccaacaa ggaaatcctc

2280

gatgaagcct acgtgatggc cagcgtggac aacccccacg tgtgccgcct gctgggcatc

2340

tgcctcacct ccaccgtgca gctcatcacg cagctcatgc ccttcggctg cctcctggac

2400

tatgtccggg aacacaaaga caatattggc tcccagtacc tgctcaactg gtgtgtgcag

2460

atcgcaaagg gcatgaacta cttggaggac cgtcgcttgg tgcaccgcga cctggcagcc

2520

aggaacgtac tggtgaaaac accgcagcat gtcaagatca cagattttgg gctggccaaa

2580

ctgctgggtg cggaagagaa agaataccat gcagaaggag gcaaagtgcc tatcaagtgg

2640

atggcattgg aatcaatttt acacagaatc tatacccacc agagtgatgt ctggagctac

2700

ggggtgactg tttgggagtt gatgaccttt ggatccaagc catatgacgg aatccctgcc

2760

agcgagatct cctccatcct ggagaaagga gaacgcctcc ctcagccacc catatgtacc

2820

atcgatgtct acatgatcat ggtcaagtgc tggatgatag acgcagatag tcgcccaaag

2880

ttccgtgagt tgatcatcga attctccaaa atggcccgag acccccagcg ctaccttgtc

2940

attcagggg atgaaagaat gcatttgcca agtcctacag actccaactt ctaccgtgcc

3000

ctgatggatg aagaagacat ggacgacgtg gtggatgccg acgagtacct catcccacag

3060

cagggcttct tcagcagccc ctccacgtca cggactcccc tcctgagctc tctgagtgca

3120

accagcaaca attccaccgt ggcttgcatt gatagaaatg ggctgcaaag ctgtcccatc

3180

aaggaagaca gcttcttgca gcgatacagc tcagacccca caggcgcctt gactgaggac

3240

agcatagacg acaccttcct cccagtgcct gaatacataa accagtccgt tcccaaaagg

3300

cccgctggct ctgtgcagaa tcctgtctat cacaatcagc ctctgaaccc cgcgcccagc

3360

agagacccac actaccagga cccccacagc actgcagtgg gcaaccccga gtatctcaac

3420

actgtccagc ccacctgtgt caacagcaca ttcgacagcc ctgcccactg ggcccagaaa

3480

ggcagccacc aaattagcct ggacaaccct gactaccagc aggacttctt tcccaaggaa

3540

gccaagccaa atggcatctt taagggctcc acagctgaaa atgcagaata cctaagggtc

3600

gcgccacaaa gcagtgaatt tattggagca

3630

<210> 14

<211> 1210

<212>PRT

<213> Homo sapiens

<400> 14

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

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

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

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

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

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

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

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

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

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

370 375 380

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

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

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

515 520 525

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

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

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

645 650 655

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

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

690 695 700

Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

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

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

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

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe

1010 1015 1020

Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu

1025 1030 1035

Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn

1040 1045 1050

Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg

1055 1060 1065

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

1070 1075 1080

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro

1085 1090 1095

Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln

1100 1105 1110

Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro

1115 1120 1125

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln

1130 1135 1140

Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala

1145 1150 1155

Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln

1160 1165 1170

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

1175 1180 1185

Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

1190 1195 1200

Ser Ser Glu Phe Ile Gly Ala

1205 1210

<210> 15

<211> 327

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

A27

VL

<400> 15

gaaatagtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc

60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa

120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcaggggccac tggcatccca

180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag

240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctcc gtggacgttc

300

ggccaaggga ccaaggtgga aatcaaa

327

<210> 16

<211> 109

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A27 VL

<400> 16

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

<210> 17

<211> 12

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A27 LCDR1

<400> 17

Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210> 18

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A27 LCDR2

<400> 18

Gly Ala Ser Ser Arg Ala Thr

15

<210> 19

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A27 LCDR3

<400> 19

Gln Gln Tyr Gly Ser Ser Pro Pro Trp Thr

1 5 10

<210> 20

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

PSM4072 VH

<400> 20

gaggtgcagc tggtggagac tgggggaggc ttggtgcaac ctggggggtc cctgagactc

60

tcctgtacag cctctggatt cacctttgaa agtcatgcca tgtactgggt ccggcaagct

120

ccagggaagg ggctggagtg ggtctcgggt attagtaatg gaggtagtag cacagagtac

180

gcagactccg tgaggggccg gttcaccatt tccagagaca attccaagaa tacggtgtat

240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gagaccaaga

300

tctccatatc tcttctacga tgctgctggt gtctggggcc aagggaccct ggtcaccgtc

360

tcctca

366

<210> 21

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of PSM4072 VH

<400> 21

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 Ala Ser Gly Phe Thr Phe Glu Ser His

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of PSM4072 HCDR1

<400> 22

Ser His Ala Met Tyr

15

<210> 23

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of PSM4072 HCDR2

<400> 23

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

1 5 10 15

Gly

<210> 24

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of PSM4072 HCDR3

<400> 24

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

1 5 10

<210> 25

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

R327

VH

<400> 25

gaggtgcagc tggtggagac cgggggaggc ttggtacagc ctggggggtc cctgagactc

60

tcctgtgcag cctctggatt cagctttgac aagtatacca 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 aggggacaat ggtcaccgtc

360

tcttca

366

<210> 26

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of R327 VH

<400> 26

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 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 Met Val Thr Val Ser Ser

115 120

<210> 27

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of R327 HCDR1

<400> 27

Lys Tyr Thr Met Asn

15

<210> 28

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of R327 HCDR2

<400> 28

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

1 5 10 15

Gly

<210> 29

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of R327 HCDR3

<400> 29

Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val

1 5 10

<210> 30

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

TfR1071 VH

<400> 30

tacgtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc

60

tcctgtgcag cctctggatt cagctttgac aagtatacca 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

<210> 31

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR1071 VH

<400> 31

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

<210> 32

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR1071 HCDR1

<400> 32

Lys Tyr Thr Met Asn

15

<210> 33

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR1071 HCDR2

<400> 33

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

1 5 10 15

Gly

<210> 34

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR1071 HCDR3

<400> 34

Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val

1 5 10

<210> 35

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

TfR4016 VH

<400> 35

tacgtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc

60

tcctgtgcag cctctggatt cagctttgac aagtatacca 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 aggggaccac ggtcaccgtc

360

tcctca

366

<210> 36

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR4016 VH

<400> 36

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 Thr Val Thr Val Ser Ser

115 120

<210> 37

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR4016 HCDR1

<400> 37

Lys Tyr Thr Met Asn

15

<210> 38

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR4016 HCDR2

<400> 38

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

1 5 10 15

Gly

<210> 39

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR4016 HCDR3

<400> 39

Ala Ser Gln Pro Trp Leu Tyr Arg Thr Gly Ala Asp Val

1 5 10

<210> 40

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

cyno186 VH

<400> 40

caggtgcagc tggtggagtc tgggggagaa ttcgtacagc ctggggggtc cctgagactc

60

tcctgtgcag cctctggatt cccctttaaa ggctatgcca tgaattgggt ccgccaggct

120

ccagggaagg gattggagtg ggtctcaaga ataagtaatg gtggtagcta catagactac

180

gcagactccg taaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat

240

ttgcaaatag acagcctgag aaccgaggac acggccgtat attactgtgc gaaagcgaag

300

gacgcctata ggtggaacaa cgctatagac gaatggggcc aagggaccct ggtcaccgtc

360

tcctca

366

<210> 41

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno186 VH

<400> 41

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Lys Gly Tyr

20 25 30

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

35 40 45

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

85 90 95

Ala Lys Ala Lys Asp Ala Tyr Arg Trp Asn Asn Ala Ile Asp Glu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 42

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno186 HCDR1

<400> 42

Gly Tyr Ala Met Asn

15

<210> 43

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno186 HCDR2

<400> 43

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

1 5 10 15

Gly

<210> 44

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno186 HCDR3

<400> 44

Ala Lys Asp Ala Tyr Arg Trp Asn Asn Ala Ile Asp Glu

1 5 10

<210> 45

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

cyno292 VH

<400> 45

caggtgcagc tggtgcaatc tgggggaggc ctggtcaagc ctggggggtc cctgagaatc

60

tcctgtacaa cctctggatt cccctttgat ggctatacca tgacctgggt ccgccaggct

120

ccagggaagg ggctggagtg ggtctcaagt attagtaatg gtggtagcac cattttcgtc

180

tcagactcag tgagaggccg attcaccatc tccagagaca acgccaagaa ttcactgtat

240

ctccaaatga acaacgtggg agccgaggac acggctgtgt attactgtgc gagagctcga

300

gatgcgtatg gctggacgct tccttctgac atctggggcc aagggacaat ggtcaccgtc

360

tcttca

366

<210> 46

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno292 VH

<400> 46

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

1 5 10 15

Ser Leu Arg Ile Ser Cys Thr Thr Ser Gly Phe Pro Phe Asp Gly Tyr

20 25 30

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

35 40 45

Ser Ser Ile Ser Asn Gly Gly Ser Thr Ile Phe Val Ser Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Asn Val Gly Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Arg Asp Ala Tyr Gly Trp Thr Leu Pro Ser Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 47

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno292 HCDR1

<400> 47

Gly Tyr Thr Met Thr

15

<210> 48

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno292 HCDR2

<400> 48

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

1 5 10 15

Gly

<210> 49

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno292 HCDR3

<400> 49

Ala Arg Asp Ala Tyr Gly Trp Thr Leu Pro Ser Asp Ile

1 5 10

<210> 50

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR1007 VH

<400> 50

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

<210> 51

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of cyno163 VH

<400> 51

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 52

<211> 123

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of 22-30 VH

<400> 52

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

Arg Glu Ala Gly Tyr Tyr Asp Ser Ser Gly Tyr Tyr Gly Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 53

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of 22-30 VL

<400> 53

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Asn Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 54

<211> 114

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of T14 VH

<400> 54

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 Val Val Ser Gly Gly Thr Phe Ser Asn Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Ser Ser Ser Trp Tyr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 55

<211> 123

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR434 VH

<400> 55

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 Tyr Asp Gly Ser Ser 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

<210> 56

<211> 123

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR435 VH

<400> 56

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

<210> 57

<211> 111

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR434,435 VL

<400> 57

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

<210> 58

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

E08

VH

<400> 58

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc

60

tcctgtgcag cctctggatt cagctttaac aacaatgcca tgaactgggt ccgtcaggct

120

ccagggaagg ggctggagtg ggtctcaggt attcgtgccg atggcggtac gacatactac

180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacactgtat

240

ctgcaaatga acagcctgag agccgaggac acggccgtat atttctgtgg aaagacgcct

300

gactgggaaa tcttctacta cgctatggac gcctggggcc aagggaccac ggtcaccgtc

360

tcctca

366

<210> 59

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E08 VH

<400> 59

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Asn Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Lys Thr Pro Asp Trp Glu Ile Phe Tyr Tyr Ala Met Asp Ala Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 60

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E08 HCDR1

<400> 60

Asn Asn Ala Met Asn

15

<210> 61

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E08 HCDR2

<400> 61

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

1 5 10 15

Gly

<210> 62

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E08 HCDR3

<400> 62

Thr Pro Asp Trp Glu Ile Phe Tyr Tyr Ala Met Asp Ala

1 5 10

<210> 63

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

E12

VH

<400> 63

caggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc

60

tcctgtgcag cctctagatt cacctttagc gcctatgcca tgggctgggt gcgccaggct

120

ccagggaagg ggctggagtg ggtctcaatt attgatagtg gtggtgtgta cacatactac

180

gcagactcca tgaagggccg cttcaccatc tccagagaca attccaagaa cacgctgtat

240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gagagatcat

300

tatggtgttg tttggggact tggagattac tggggccagg gaaccctggt cactgtctcc

360

tca

363

<210> 64

<211> 121

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E12 VH

<400> 64

Gln 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 Arg Phe Thr Phe Ser Ala Tyr

20 25 30

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

35 40 45

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

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 Arg Asp His Tyr Gly Val Val Trp Gly Leu Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 65

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E12 HCDR1

<400> 65

Ala Tyr Ala Met Gly

15

<210> 66

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E12 HCDR2

<400> 66

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

1 5 10 15

Gly

<210> 67

<211> 12

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E12 HCDR3

<400> 67

Asp His Tyr Gly Val Val Trp Gly Leu Gly Asp Tyr

1 5 10

<210> 68

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

E17

VH

<400> 68

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac tctgtccctc

60

acctgcgcac tgtacagtgg gtccttcagt gctcaggact ggagctggat ccgccagtcc

120

ccagagaagg ggctggagtg gattggggaa atctggcaag ggggaaaaac caactacaac

180

ccgtccctca agagtcgagt tagtatatca agagacaact ccaagaacca gttgtccctg

240

cagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag ggtggactgg

300

tcttatcgtg tttttgaaat ctggggccaa gggacaatgg tcaccgtctc ttca

354

<210> 69

<211> 118

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E17 VH

<400> 69

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

1 5 10 15

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

20 25 30

Asp Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Trp Gln Gly Gly Lys Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Met Val Thr Val Ser Ser

115

<210> 70

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E17 HCDR1

<400> 70

Ala Gln Asp Trp Ser

15

<210> 71

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E17 HCDR2

<400> 71

Glu Ile Trp Gln Gly Gly Lys Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 72

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of E17 HCDR3

<400> 72

Val Asp Trp Ser Tyr Arg Val Phe Glu Ile

1 5 10

<210> 73

<211> 123

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of KME07 VH

<400> 73

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 Ser Ser Phe

20 25 30

Ala Phe Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Val Arg Pro Gly Phe Gly Thr Glu Ile Leu Thr Gln Asn Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Thr Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Leu Leu Arg Gly Gly Tyr Ile Glu Asn Pro Phe Glu Ile

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 74

<211> 123

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of KME09 VH

<400> 74

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 Ser Ser Phe

20 25 30

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

35 40 45

Gly Glu Ile Arg Pro Gly Phe Gly Thr Glu Ile Tyr Ala Gln Asn Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 75

<211> 125

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of KME11 VH

<400> 75

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 Leu Ser Thr Tyr

20 25 30

Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Phe Ala Trp Met

35 40 45

Gly Glu Ile Ile Pro Ser Leu Gly Ile Val Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Gly Asp Glu Leu Ser Glu Gly His Ser Glu Tyr Tyr His Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 76

<211> 119

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Cetuximab VH

<400> 76

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 77

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Cetuximab VL

<400> 77

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

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 78

<211> 117

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of HN3 VH

<400> 78

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

<210> 79

<211> 2

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid of

sequence of linker

<400> 79

Glu Ser

1

<210> 80

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid of

sequence of linker

<400> 80

Glu Ser Lys Tyr Gly

15

<210> 81

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid of

sequence of linker

<400> 81

Glu Ser Lys Tyr Gly Pro Pro

15

<210> 82

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid of

sequence of linker

<400> 82

Gly Gly Gly Gly Ser

15

<210> 83

<211> 981

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

IgG4PE R409K constant region

<400> 83

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 ccadgaagac 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

tggggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc

840

gacggctcct tcttcctcta cagcaagcta accgtggaca agagcaggtg gcaggagggg

900

aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc

960

ctctccctgt ctctgggtaa a

981

<210> 84

<211> 327

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of IgG4PE R409K constant region

<400> 84

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 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

<210> 85

<211> 294

<212> DNA

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the base sequence of

modified codons of IgG4 CH1

<400> 85

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 ggtc

294

<210> 86

<211> 327

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of IgG4PE constant region

<400> 86

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 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

Arg 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

<210> 87

<211> 763

<212>PRT

<213> Mus musculus

<400> 87

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

<210> 88

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Apical domain mouse chimera of human TfR extracellular

domain

<400> 88

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

<210> 89

<211> 675

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Protease-like domain mouse chimera of human TfR

extracellular

domain

<400> 89

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

<210> 90

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A01

<400> 90

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 Lys Asn 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 Ser 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

<210> 91

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A02

<400> 91

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

<210> 92

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A03

<400> 92

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

<210> 93

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A04

<400> 93

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

<210> 94

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A05

<400> 94

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

<210> 95

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A06

<400> 95

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 Gln 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

<210> 96

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A07

<400> 96

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

<210> 97

<211> 671

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A08

<400> 97

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 Lys Asn Gly Arg Leu Asp Leu Val Glu Asn Pro Gly

115 120 125

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

130 135 140

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

145 150 155 160

Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe Ala

165 170 175

Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu Ile

180 185 190

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

195 200 205

Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe

210 215 220

Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly Leu

225 230 235 240

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

245 250 255

Phe Gly Lys Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp Ser

260 265 270

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

275 280 285

Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly Val Ile

290 295 300

Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala Gln Arg

305 310 315 320

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

325 330 335

Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys Asp Gly

340 345 350

Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala Gly Asp

355 360 365

Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser Ser

370 375 380

Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala Val Leu

385 390 395 400

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

405 410 415

Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly Gln Phe

420 425 430

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

435 440 445

Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala Val

450 455 460

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

465 470 475 480

Met Asp Thr Tyr Lys Glu Leu Ile Glu Arg Ile Pro Glu Leu Asn Lys

485 490 495

Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile Lys Leu

500 505 510

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

515 520 525

Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp Ile Lys

530 535 540

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

545 550 555 560

Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala Glu Lys

565 570 575

Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met Arg Val

580 585 590

Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser Pro Phe

595 600 605

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

610 615 620

Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn Glu Thr

625 630 635 640

Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Ala

645 650 655

Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu Phe

660 665 670

<210> 98

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A09

<400> 98

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 Pro Thr Glu Val Ser 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

<210> 99

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A10

<400> 99

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 Pro Thr Glu 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

<210> 100

<211> 673

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A11

<400> 100

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 Ser

100 105 110

Val Ile Ile Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn

115 120 125

Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys

130 135 140

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

145 150 155 160

Thr Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr

165 170 175

Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val

180 185 190

Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu

195 200 205

Ser Phe Phe Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro

210 215 220

Gly Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser

225 230 235 240

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

245 250 255

Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr

260 265 270

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

275 280 285

Thr Val Lys Asn Val Leu Lys Glu Ile Lys Ile Leu Asn Ile Phe Gly

290 295 300

Val Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val Val Gly Ala

305 310 315 320

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

325 330 335

Ala Leu Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met Val Leu Lys

340 345 350

Asp Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser Trp Ser Ala

355 360 365

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

370 375 380

Ser Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn Leu Asp Lys Ala

385 390 395 400

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

405 410 415

Thr Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro Val Thr Gly

420 425 430

Gln Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val Glu Lys Leu

435 440 445

Thr Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser Gly Ile Pro

450 455 460

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

465 470 475 480

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

485 490 495

Asn Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln Phe Val Ile

500 505 510

Lys Leu Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu Arg Tyr Asn

515 520 525

Ser Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr Arg Ala Asp

530 535 540

Ile Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly

545 550 555 560

Asp Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp Phe Gly Asn Ala

565 570 575

Glu Lys Thr Asp Arg Phe Val Met Lys Lys Leu Asn Asp Arg Val Met

580 585 590

Arg Val Glu Tyr His Phe Leu Ser Pro Tyr Val Ser Pro Lys Glu Ser

595 600 605

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

610 615 620

Leu Leu Glu Asn Leu Lys Leu Arg Lys Gln Asn Asn Gly Ala Phe Asn

625 630 635 640

Glu Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln

645 650 655

Gly Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile Asp Asn Glu

660 665 670

Phe

<210> 101

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A12

<400> 101

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

Glu 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 Ala

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

<210> 102

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A13

<400> 102

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 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

<210> 103

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A14

<400> 103

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

<210> 104

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A15

<400> 104

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 Met Val

100 105 110

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

<210> 105

<211> 672

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of A16

<400> 105

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 Met 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 Glu 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 Ile

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

<210> 106

<211> 122

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of VH starting with TfR1071 EVQL

<400> 106

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

<210> 107

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR1007 HCDR1

<400> 107

Gly Tyr Ser Met Ser

15

<210> 108

<211> 17

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR1007 HCDR2

<400> 108

Ser Leu Ser Asn Gly Gly Gly Ser Ser Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 109

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of TfR1007 HCDR3

<400> 109

Thr Leu Gly Ala Tyr Tyr Ile Lys Ser Ala Met Asp Val

1 5 10

<210> 110

<211> 119

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Cetuximab VH

<400> 110

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 111

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Cetuximab VL

<400> 111

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

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 112

<211> 119

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Panitumumab VH

<400> 112

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

1 5 10 15

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

20 25 30

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

35 40 45

Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 113

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Panitumumab VL

<400> 113

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu

85 90 95

Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 114

<211> 121

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Necitumumab VH

<400> 114

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

1 5 10 15

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

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 115

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Necitumumab VL

<400> 115

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Gly Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Ala Glu Ile Lys

100 105

<210> 116

<211> 123

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Nimotuzumab VH

<400> 116

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

Gly Gly Ile Asn Pro Thr Ser Gly Gly Ser Asn Phe Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Gln Gly Leu Trp Phe Asp Ser Asp Gly Arg Gly Phe Asp Phe

100 105 110

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 117

<211> 112

<212>PRT

<213> Artificial Sequence

<220>

<223> description of the artificial sequence: the amino acid

sequence

of Nimotuzumab VL

<400> 117

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Asn Leu Leu Ile Tyr Lys Val Ser Asn Arg Glu Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Tyr

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<---

Claims (25)

1. A bispecific antibody or fragment of a bispecific antibody that binds to a transferrin receptor (TfR) and a cell surface antigen, comprising an IgG portion that binds to the TfR and an N-terminal polypeptide that binds to a cell surface antigen, wherein: (i) said cell surface antigen is EGFR; (ii) The IgG portion includes one heavy chain variable region (VH) selected from the following (ai)-(ci), and a light chain variable region (VL) having CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 17-19 respectively: (ai) VH having CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 32-34, respectively; (bi) VH having CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 42-44, respectively; And (ci) VH having CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 47-49, respectively; (iii) the N-terminal side polypeptide is an anti-EGFR Fab antibody, wherein the antibody comprises a VH having CDRs 1-3 containing the amino acid sequences represented by SEQ ID NOs: 60-62, respectively, or SEQ ID NOs: 65-67, respectively , and VL having CDRs 1-3 containing the amino acid sequences represented by SEQ ID NO: 17-19, respectively; And (iv) said N-terminal side polypeptide is linked directly or via a linker structure to the N-terminus of the heavy chain of said IgG portion. 2. A bispecific antibody or fragment of a bispecific antibody according to claim 1, wherein said bispecific antibody binds bivalently to both TfR and EGFR. 3. A bispecific antibody or fragment of a bispecific antibody according to claim 1 or 2, wherein the IgG portion comprises a VH containing the amino acid sequence represented by any of the sequences of SEQ ID NOs: 31, 41 and 46, and a VL containing the amino acid sequence represented by SEQ ID NO: 16. 4. A bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-3, wherein the IgG portion contains a heavy chain constant region of the amino acid sequence represented by SEQ ID NO: 84 or SEQ ID NO: 86. 5. A bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-4, wherein the anti-EGFR antibody is an antibody comprising a VH containing the amino acid sequence represented by SEQ ID NO: 59 or SEQ ID NO: 64, and a VL containing the amino acid sequence represented by SEQ ID NO: 16. 6. Bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-5, in which the C-terminus of the Fab heavy chain is directly connected to the N-terminus of the heavy chain of the IgG portion. 7. A bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-5, wherein the C-terminus of the Fab heavy chain is linked to the N-terminus of the heavy chain of the IgG portion via a linker. 8. The bispecific antibody or fragment of the bispecific antibody according to claim 7, wherein the amino acid sequence of the linker consists of all or part of the amino acid sequence of the hinge region of the IgG molecule. 9. Bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-8, in which the amino acid sequence that makes up the light chain in the Fab fragment and the amino acid sequence that makes up the light chain in the IgG part are the same. 10. DNA encoding a bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-9. 11. Recombinant expression vector containing DNA according to claim 10. 12. A transformant for obtaining a bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-9, containing the recombinant vector according to claim 11, where the transformant is a cell. 13. A method for producing a bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-9, including cultivating the transformant according to claim 12 in a culture medium that ensures the transformant produces and accumulates in the culture a bispecific antibody or a fragment of this bispecific antibody according to any one of claims. 1-9, and also including the isolation of the specified bispecific antibody or a fragment of this bispecific antibody from this culture. 14. A therapeutic agent used for a disease associated with at least one of human TfR or EGFR, wherein said agent comprises as an active ingredient a bispecific antibody or a fragment of this bispecific antibody according to any one of claims. 1-9, wherein the disease associated with at least one of human TfR or EGFR is a cancer. 15. A therapeutic method for treating a disease associated with at least one of human TfR and EGFR, which uses a bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-9, wherein the disease associated with at least one of human TfR or EGFR is a cancer. 16. A bispecific antibody or a fragment of this bispecific antibody according to any one of paragraphs. 1-9 for use in the treatment of a disease associated with at least one of human TfR and EGFR, wherein the disease associated with at least one of human TfR or EGFR is a cancer. 17. The use of a bispecific antibody or a fragment of a bispecific antibody according to any one of paragraphs. 1-9 for the preparation of a therapeutic agent for treating a disease associated with at least one of human TfR and EGFR, wherein the disease associated with at least one of human TfR or EGFR is a cancer. 18. The use of a bispecific antibody or a fragment of a bispecific antibody according to any one of paragraphs. 1-9 for the preparation of a therapeutic agent for treating a disease associated with at least one of human TfR and EGFR, wherein the disease associated with at least one of human TfR or EGFR is a cancer.