RU2776821C2 - Monoclonal antibody to nav1.7 - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a monoclonal antibody or its fragment, which specifically binds to Nav1.7. A pharmaceutical composition containing the specified antibody, polynucleotide encoding the specified antibody, and an expression vector containing the specified polynucleotide are also disclosed.

EFFECT: invention allows for the effective treatment of diseases associated with Nav1.7 factor.

9 cl, 5 dwg, 5 tbl, 7 ex

Description

[Technical field]

[0001]

The present invention relates to a novel anti-Nav1.7 monoclonal antibody or fragment thereof. More specifically, the present invention relates to an antibody or fragment thereof that specifically binds to Nav1.7. More specifically, the present invention relates to a monoclonal antibody or fragment thereof that selectively inhibits Nav1.7, or an antibody fragment, or a pharmaceutical composition containing such an antibody, or a Nav1.7 detection kit.

[prior art]

[0002]

Nav1.7 is a voltage-gated sodium ion channel that is encoded by the SCN9A gene and is predominantly expressed in peripheral nerves (non-patent literature: 1). Nav1.7 contains four domains A, B, C and D, each of six transmembrane protein helices (S1, S2, S3, S4, S5 and S6) and three extracellular hydrophilic loops E1, E2 and E3.

[0003]

Inflammatory pain is known to be reduced in the Nav1.7 knockout mouse (non-patent literature: 2). The Nav1.7 inhibitor, which is a small molecular weight compound, has been confirmed to be effective in erythromelalgia (non-patent literature: 3), trigeminal neuralgia (non-patent literature: 4), and the like.

[0004]

An anti-Nav1.7 monoclonal antibody that is effective for pain (pain reliever) and pruritus (antipruritus) is described in Patent Literature: 1 to 7, etc. An antibody that binds to the extracellular region of E3 domain C is described in patent literature: 1 to 3.

[Citation List]

[Patent Literature]

[0005]

[Patent literature: 1] WO2011/051350

[Patent literature: 2] US8734798

[Patent literature: 3] US8986954

[Patent literature: 4] US9266953

[Patent literature: 5] WO2014/159595

[Patent literature: 6] WO2015/032916

[Patent literature: 7] WO2015/035173

[Non-Patent Literature]

[0006]

[Non-Patent Literature: 1] PNAS(1997)94:1527-1532

[Non-Patent Document: 2] PNAS(2004)101:12706-12711

[Non-Patent Document: 3] Pain(2012)153(1):80-85

[Non-Patent Document: 4] THE LANCET NEUROLOGY(2017)16(4):291-300

[Summary of the Invention]

[Problem to be solved by invention]

[0007]

It is an object of the present invention to provide a novel anti-Nav1.7 monoclonal antibody or antibody fragment that can be used as a therapeutic agent for pain, itching, and the like.

[Solution]

[0008]

The inventors of the present invention have made extensive studies and have found a monoclonal antibody that specifically binds to the extracellular region of E3 of the C domain of Nav1.7 and selectively inhibits Nav1.7. We also found that the monoclonal antibody of the present invention has an analgesic or antipruritic inhibitory effect.

[0009] Specifically, the present invention relates to:

(1-1)

A monoclonal antibody or fragment thereof that binds to Nav1.7,

having

a1) heavy chain variable region,

containing a CDR1 with the amino acid sequence from SEQ ID NO: 27, a CDR2 with the amino acid sequence from SEQ ID NO: 28 and a CDR3 with the amino acid sequence from SEQ ID NO: 29, where one or two amino acids can be deleted, substituted, inserted and/or added , and

light chain variable region

containing a CDR1 with the amino acid sequence from SEQ ID NO: 34, a CDR2 with the amino acid sequence from SEQ ID NO: 35 and a CDR3 with the amino acid sequence from SEQ ID NO: 50, where one or two amino acids can be deleted, substituted, inserted and/or added ;

(i.e., heavy and light chain variable regions containing CDRs from 12H4, h12H4, h12H4-2 and h12H4-3 according to the example, or variants thereof),

heavy chain variable region containing

b1-1) CDR1 with the amino acid sequence of SEQ ID NO: 3, CDR2 with the amino acid sequence of SEQ ID NO: 4 and CDR3 with the amino acid sequence of SEQ ID NO: 5, where one or two amino acids can be deleted, substituted, inserted and /or added,

b1-2) CDR1 with the amino acid sequence of SEQ ID NO: 15, CDR2 with the amino acid sequence of SEQ ID NO: 16 and CDR3 with the amino acid sequence of SEQ ID NO: 17, where one or two amino acids can be deleted, substituted, inserted and /or added,

b1-3) CDR1 with the amino acid sequence of SEQ ID NO: 15, CDR2 with the amino acid sequence of SEQ ID NO: 24 and CDR3 with the amino acid sequence of SEQ ID NO: 25, where one or two amino acids can be deleted, substituted, inserted and /or added, or

b1-4) CDR1 with the amino acid sequence of SEQ ID NO: 21, CDR2 with the amino acid sequence of SEQ ID NO: 22 and CDR3 with the amino acid sequence of SEQ ID NO: 17, where one or two amino acids can be deleted, substituted, inserted and /or added, and

light chain variable region

containing a CDR1 with the amino acid sequence from SEQ ID NO: 34, a CDR2 with the amino acid sequence from SEQ ID NO: 35 and a CDR3 with the amino acid sequence from SEQ ID NO: 36, where one or two amino acids can be deleted, substituted, inserted and/or added ;

(i.e., heavy and light chain variable regions containing CDRs from 3B2, 3B2/15C8, 15C8, 29G3, or 28B5/15C8 according to the example, or variants thereof)

c1) heavy chain variable region,

containing a CDR1 with the amino acid sequence from SEQ ID NO: 7, a CDR2 with the amino acid sequence from SEQ ID NO: 8 and a CDR3 with the amino acid sequence from SEQ ID NO: 9, where one or two amino acids can be deleted, substituted, inserted and/or added , and

light chain variable region

containing CDR1 with the amino acid sequence from SEQ ID NO: 38, CDR2 with the amino acid sequence from SEQ ID NO: 39 and CDR3 with the amino acid sequence from SEQ ID NO: 40, where one or two amino acids can be deleted, substituted, inserted and/or added ;

(i.e., heavy and light chain variable regions containing CDRs from 5E12 according to the example, or variants thereof)

d1) heavy chain variable region,

containing a CDR1 with the amino acid sequence from SEQ ID NO: 11, a CDR2 with the amino acid sequence from SEQ ID NO: 12 and a CDR3 with the amino acid sequence from SEQ ID NO: 13, where one or two amino acids can be deleted, substituted, inserted and/or added , and

light chain variable region

containing a CDR1 with the amino acid sequence from SEQ ID NO: 38, a CDR2 with the amino acid sequence from SEQ ID NO: 42 and a CDR3 with the amino acid sequence from SEQ ID NO: 40, where one or two amino acids can be deleted, substituted, inserted and/or added ;

(i.e., heavy and light chain variable regions containing CDRs from 7B9 according to the example, or variants thereof)

e1) heavy chain variable region

containing a CDR1 with the amino acid sequence from SEQ ID NO: 15, a CDR2 with the amino acid sequence from SEQ ID NO: 19 and a CDR3 with the amino acid sequence from SEQ ID NO: 17, where one or two amino acids can be deleted, substituted, inserted and /or added, and

light chain variable region

containing a CDR1 with the amino acid sequence of SEQ ID NO: 34, a CDR2 with the amino acid sequence of SEQ ID NO: 45 and a CDR3 with the amino acid sequence of SEQ ID NO: 36, where one or two amino acids can be deleted, substituted, inserted and /or added;

(i.e., heavy and light chain variable regions containing CDRs from 15H6 according to the example, or variants thereof),

heavy chain variable region containing

f1-1) CDR1 with the amino acid sequence of SEQ ID NO: 21, CDR2 with the amino acid sequence of SEQ ID NO: 22 and CDR3 with the amino acid sequence of SEQ ID NO: 17, where one or two amino acids can be deleted, substituted, inserted and /or added, or

f1-2) CDR1 with the amino acid sequence of SEQ ID NO: 3, CDR2 with the amino acid sequence of SEQ ID NO: 4 and CDR3 with the amino acid sequence of SEQ ID NO: 5, where one or two amino acids can be deleted, substituted, inserted and /or added, and

light chain variable region

containing a CDR1 with the amino acid sequence from SEQ ID NO: 34, a CDR2 with the amino acid sequence from SEQ ID NO: 35 and a CDR3 with the amino acid sequence from SEQ ID NO: 47, where one or two amino acids can be deleted, substituted, inserted and/or added ;

(i.e., heavy and light chain variable regions containing CDRs from 28B5 or 3B2/28B5 according to the example, or variants thereof),

g1) heavy chain variable region,

containing a CDR1 with the amino acid sequence from SEQ ID NO: 31, a CDR2 with the amino acid sequence from SEQ ID NO: 32 and a CDR3 with the amino acid sequence from SEQ ID NO: 29, where one or two amino acids can be deleted, substituted, inserted and/or added , and

light chain variable region

containing a CDR1 with the amino acid sequence from SEQ ID NO: 34, a CDR2 with the amino acid sequence from SEQ ID NO: 35 and a CDR3 with the amino acid sequence from SEQ ID NO: 50, where one or two amino acids can be deleted, substituted, inserted and/or added ;

(i.e., heavy and light chain variable regions containing CDRs from 22D3 according to the example, or variants thereof),

h1) heavy chain variable region,

containing CDR1 with the amino acid sequence from SEQ ID NO: 56: G-Y-Y-Xaa4-H (where Xaa4 is M or I), CDR2 with the amino acid sequence from SEQ ID NO: 57: L-I-I-P-Y-Xaa5-G-Xaa6-Xaa7-F-Y-N- Xaa8-K-F-Xaa9-G (where Xaa5 is S or N, Xaa6 is D or E, Xaa7 is T or I, Xaa8 is Q or P, and Xaa9 is K or R) and CDR3 with the amino acid sequence of SEQ ID NO: 58: A-Xaa10-V-S-Y-A-M-D-Y (wherein Xaa10 is E or D) wherein one or two amino acids may be deleted, substituted, inserted and/or added, and

light chain variable region

containing a CDR1 with the amino acid sequence from SEQ ID NO: 34, a CDR2 with the amino acid sequence from SEQ ID NO: 54: K-V-S-N-R-Xaa1-S (where Xaa1 is F or I) and a CDR3 with the amino acid sequence from SEQ ID NO: 55: S-Q-S -Xaa2-H-V-P-Xaa3-T (wherein Xaa2 is T or I and Xaa3 is F or W), where one or two amino acids can be deleted, substituted, inserted and/or added.

(ie, heavy and light chain variable regions containing CDRs with the consensus sequences of multiple antibodies according to Example 3, or variants thereof).

[0010]

(1-2) An antibody or antibody fragment according to (1-1),

having

heavy chain variable region containing

CDR1 with the amino acid sequence of SEQ ID NO: 27, where one or two amino acids can be deleted, substituted, inserted and/or added at any position other than position 10;

CDR2 with the amino acid sequence of SEQ ID NO: 28, where one or two amino acids can be deleted, substituted, inserted and/or added, and CDR3 with the amino acid sequence of SEQ ID NO: 29, where one or two amino acids can be deleted, substituted , inserted and/or added at any position other than position 2 and 6, and

light chain variable region

containing a CDR1 with the amino acid sequence from SEQ ID NO: 34, where one or two amino acids can be deleted, substituted, inserted and/or added, a CDR2 with an amino acid sequence from SEQ ID NO: 35, where one or two amino acids can be deleted, substituted , inserted and/or added, and a CDR3 with the amino acid sequence of SEQ ID NO: 50, where one or two amino acids may be deleted, substituted, inserted and/or added at any position other than position 5. (i.e., heavy and light chain variable regions containing CDRs in which critical sequences in 12H4 are required according to Example 6, or variants thereof)

[0011]

(2) An antibody or antibody fragment according to (1-1) or (1-2),

having

a1) heavy chain variable region,

containing CDR1 with the amino acid sequence from SEQ ID NO: 27, CDR2 with the amino acid sequence from SEQ ID NO: 28 and CDR3 with the amino acid sequence from SEQ ID NO: 29, and

light chain variable region

containing CDR1 with the amino acid sequence of SEQ ID NO: 34, CDR2 with the amino acid sequence of SEQ ID NO: 35 and CDR3 with the amino acid sequence of SEQ ID NO: 50;

(i.e., heavy and light chain variable regions containing CDRs from 12H4, h12H4, h12H4-2 and h12H4-3 according to the example),

heavy chain variable region containing

b1-1) CDR1 with the amino acid sequence of SEQ ID NO: 3, CDR2 with the amino acid sequence of SEQ ID NO: 4 and CDR3 with the amino acid sequence of SEQ ID NO: 5,

b1-2) CDR1 with the amino acid sequence of SEQ ID NO: 15, CDR2 with the amino acid sequence of SEQ ID NO: 16 and CDR3 with the amino acid sequence of SEQ ID NO: 17,

b1-3) CDR1 with the amino acid sequence of SEQ ID NO: 15, CDR2 with the amino acid sequence of SEQ ID NO: 24 and CDR3 with the amino acid sequence of SEQ ID NO: 25, or

b1-4) CDR1 with the amino acid sequence of SEQ ID NO: 21, CDR2 with the amino acid sequence of SEQ ID NO: 22 and CDR3 with the amino acid sequence of SEQ ID NO: 17, and

light chain variable region

containing CDR1 with the amino acid sequence of SEQ ID NO: 34, CDR2 with the amino acid sequence of SEQ ID NO: 35 and CDR3 with the amino acid sequence of SEQ ID NO: 36;

(i.e., heavy and light chain variable regions containing CDRs from 3B2, 3B2/15C8, 15C8, 29G3 or 28B5/15C8 according to the example)

c1) heavy chain variable region,

containing CDR1 with the amino acid sequence from SEQ ID NO: 7, CDR2 with the amino acid sequence from SEQ ID NO: 8 and CDR3 with the amino acid sequence from SEQ ID NO: 9, and

light chain variable region

containing CDR1 with the amino acid sequence of SEQ ID NO: 38, CDR2 with the amino acid sequence of SEQ ID NO: 39 and CDR3 with the amino acid sequence of SEQ ID NO: 40;

(ie, heavy and light chain variable regions containing CDRs from 5E12 according to the example);

d1) heavy chain variable region,

containing CDR1 with the amino acid sequence from SEQ ID NO: 11, CDR2 with the amino acid sequence from SEQ ID NO: 12 and CDR3 with the amino acid sequence from SEQ ID NO: 13, and

light chain variable region

containing CDR1 with the amino acid sequence of SEQ ID NO: 38, CDR2 with the amino acid sequence of SEQ ID NO: 42 and CDR3 with the amino acid sequence of SEQ ID NO: 40;

(i.e., heavy and light chain variable regions containing CDRs from 7B9 according to the example)

e1) heavy chain variable region,

containing CDR1 with the amino acid sequence from SEQ ID NO: 15, CDR2 with the amino acid sequence from SEQ ID NO: 19 and CDR3 with the amino acid sequence from SEQ ID NO: 17, and

light chain variable region

containing CDR1 with the amino acid sequence of SEQ ID NO: 34, CDR2 with the amino acid sequence of SEQ ID NO: 45 and CDR3 with the amino acid sequence of SEQ ID NO: 36;

(i.e., heavy and light chain variable regions containing CDRs from 15H6 according to the example);

heavy chain variable region containing

f1-1) CDR1 with the amino acid sequence of SEQ ID NO: 21, CDR2 with the amino acid sequence of SEQ ID NO: 22 and CDR3 with the amino acid sequence of SEQ ID NO: 17, or

f1-2) CDR1 with the amino acid sequence of SEQ ID NO: 3, CDR2 with the amino acid sequence of SEQ ID NO: 4 and CDR3 with the amino acid sequence of SEQ ID NO: 5, and

light chain variable region

containing CDR1 with the amino acid sequence of SEQ ID NO: 34, CDR2 with the amino acid sequence of SEQ ID NO: 35 and CDR3 with the amino acid sequence of SEQ ID NO: 47;

(ie, heavy and light chain variable regions containing CDRs from 28B5 or 3B2/28B5 according to the example);

g1) heavy chain variable region,

containing CDR1 with the amino acid sequence from SEQ ID NO: 31, CDR2 with the amino acid sequence from SEQ ID NO: 32 and CDR3 with the amino acid sequence from SEQ ID NO: 29, and

light chain variable region

containing CDR1 with the amino acid sequence of SEQ ID NO: 34, CDR2 with the amino acid sequence of SEQ ID NO: 35 and CDR3 with the amino acid sequence of SEQ ID NO: 50; or

(ie, heavy and light chain variable regions containing CDRs from 22D3 according to the example);

h1) heavy chain variable region,

containing CDR1 with the amino acid sequence from SEQ ID NO: 56, CDR2 with the amino acid sequence from SEQ ID NO: 57 and CDR3 with the amino acid sequence from SEQ ID NO: 58, and

light chain variable region

containing CDR1 with the amino acid sequence from SEQ ID NO: 34, CDR2 with the amino acid sequence from SEQ ID NO: 54 and CDR3 with the amino acid sequence from SEQ ID NO: 55.

(i.e., heavy and light chain variable regions containing CDRs with consensus sequences of multiple antibodies according to Example 3)

[0012]

(3) an antibody or antibody fragment according to (1-1) or (1-2),

having

a2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 52, 59 or 60 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 52, 59 or 60, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 53 or 61 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 53 or 61;

b2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 26 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 26, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 49 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 49;

c2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 23 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 23, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 48 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 48;

d2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 2 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 2, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 33 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 33;

e2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 6 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 6, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 37 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 37;

f2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 10 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 10, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 41 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 41;

g2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 14 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 14, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 43 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 43;

h2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 18 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 18, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 44 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 44;

i2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 20 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 20, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 46 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 46;

j2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 30 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 30, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 51 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 51;

k2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 2 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 2, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 46 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 46;

l2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 2 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 2, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 43 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 43; or

m2) heavy chain variable region

with an amino acid sequence from SEQ ID NO: 20 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 20, and

light chain variable region

with an amino acid sequence from SEQ ID NO: 43 or an amino acid sequence at least 95% identical to the amino acid sequence from SEQ ID NO: 43.

Specific examples of antibodies are listed in Table 1 in Example.

[0013]

(4) An antibody or antibody fragment according to (3) having

a2-1) a heavy chain variable region with the amino acid sequence of SEQ ID NO: 52, and

a light chain variable region with the amino acid sequence of SEQ ID NO: 53;

a2-2) a heavy chain variable region with the amino acid sequence of SEQ ID NO: 59, and

a light chain variable region with the amino acid sequence of SEQ ID NO: 53; or

a2-3) a heavy chain variable region with the amino acid sequence of SEQ ID NO: 60, and

a light chain variable region with the amino acid sequence of SEQ ID NO: 61.

(5) An antibody or antibody fragment according to any of (1) to (4),

additionally having

a heavy chain constant region with the amino acid sequence of SEQ ID NO: 62 or 63, and

a light chain constant region with the amino acid sequence of SEQ ID NO: 64.

Specific examples of the antibodies in (4) and (5) are listed in Table 2 in Example.

[0014]

(6) A pharmaceutical composition containing an antibody or fragment thereof according to any of (1-1), (1-2) and (2)-(5).

(7) A pharmaceutical composition according to (6) for treating or preventing pain and/or itching.

(8) A polynucleotide encoding an antibody heavy chain variable region according to (3) or (4), wherein the polynucleotide optionally encodes an antibody heavy chain constant region according to (5).

(9) A polynucleotide encoding an antibody heavy chain variable region according to (3) or (4), wherein the polynucleotide optionally encodes an antibody light chain constant region according to (5).

(10) An expression vector containing a polynucleotide according to (8) or (9).

[0015]

(11) A polynucleotide encoding an antibody or fragment thereof according to any of (1-1), (1-2), and (2)-(5).

(12) An antibody or fragment thereof according to any of (1-1), (1-2), and (2)-(5), wherein the fragment is Fab, Fab', F(ab') ₂ , scFv , dsFv or diabody.

(13) A method for preventing or treating a disease associated with Nav1.7, comprising administering a monoclonal antibody or fragment thereof according to any of (1-1), (1-2), and (2)-(5).

(14) A monoclonal antibody or fragment thereof according to any of (1-1), (1-2) and (2) to (5) for producing an agent for treating or preventing a disease associated with Nav1.7.

(15) A monoclonal antibody or fragment thereof according to any of (1-1), (1-2) and (2)-(5) for treating or preventing a disease associated with Nav1.7.

(16) The method according to (13), or the antibody or fragment thereof according to (14) or (15), wherein the disease associated with Nav1.7 is pain and/or itching.

(17) A Nav1.7 detection kit comprising a monoclonal antibody or fragment thereof according to any of (1-1), (1-2) and (2)-(5).

[Invention effect]

[0016]

Because the monoclonal antibody or fragment thereof of the present invention specifically binds to Nav1.7, it can be used to detect Nav1.7 in biological samples. In addition, since the monoclonal antibody or fragment thereof of the present invention has the property of selectively inhibiting Nav1.7, a pharmaceutical composition containing it is very useful in medicine, especially for treating or preventing a disease associated with Nav1.7.

[Brief Description of Drawings]

[0017]

[Figure 1] Kabat numbering and heavy chain alignment results from 15C8 (SEQ ID NO: 14), 15H6 (SEQ ID NO: 18), 28B5 (SEQ ID NO: 20), and 29G3 (SEQ ID NO: 23).

[Figure 2] Kabat numbering and light chain alignment results from 15C8 (SEQ ID NO: 43), light chain from 15H6 (SEQ ID NO: 44), 28B5 (SEQ ID NO: 46) and 29G3 (SEQ ID NO: 48 ).

[Figure 3] Kabat numbering of h12H4 heavy chain (SEQ ID NO:52, FIG. 3A) and h12H4 light chain (SEQ ID NO:53, FIG. 3B).

[Figure 4] Evaluation of drug efficacy by local intraplantar injection models with ligation of part of the sciatic nerve.

[Figure 5] Evaluation of the effectiveness of the drug by intravenous injection models with ligation of the part of the sciatic nerve.

[Embodiment]

[0018]

The terms in the present description refer to commonly used in this field values, unless otherwise indicated.

In the present invention, methods known in the art can be used to produce antibodies. Examples include the methods described in "Immunochemistry in Practice" (Blackwell Scientific Publications).

You can also use the methods of genetic engineering known in this field. Examples include the methods described in Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012), Current Protocols Essential Laboratory Techniques, Current Protocols (2012).

[0019]

Human Nav1.7 is a protein (UniProtKB/Swiss-Prot:Q15858) consisting of amino acids encoded by the SCN9A gene.

[0020]

A monoclonal antibody or fragment thereof in the present invention is a monoclonal antibody or fragment thereof having the CDR or heavy/light chain variable regions provided herein. The antibody or fragment thereof may be from any class (eg, IgG, IgE, IgM, IgD, or IgA) or subclass of immunoglobulin molecules and may be obtained from any species, including mouse, rat, shark, rabbit, pig, hamster, camel, llama , goat and human. A preferred antibody or fragment thereof is a humanized monoclonal antibody or fragment thereof.

[0021]

As used herein, "monoclonal antibody fragment" means a portion of a monoclonal antibody where the fragment specifically binds to Nav1.7 and selectively inhibits Nav1.7, as does said monoclonal antibody.

[0022]

Specific examples include Fab (antigen binding fragment), Fab', F(ab') ₂ , single chain antibody (single chain Fv; also referred to herein as scFv), disulfide-stabilized antibody (disulfide-stabilized Fv; hereinafter, referred to as dsFv), dimerized fragments of the V region (hereinafter referred to as diatelo) and peptides containing CDRs that specifically bind to human Nav1.7 (Expert Opinion on Therapeutic Patents, Vol. 6, No. 5, pp 441-456, 1996).

[0023]

Fab is an antibody fragment having a molecular weight of approximately 50,000 with antigen binding activity and consisting of approximately half of the N-terminal region of the H chain and the entire L chain, obtained by breaking with the papain enzyme the peptide portion of the upper part of two disulfide bonds (SS- ties) that crosslink two H chains in the IgG hinge region. The Fab used in the present invention can be obtained by treating the monoclonal antibody of the present invention with papain. Alternatively, the Fab can be obtained by inserting the DNA encoding the Fab from the monoclonal antibody of the present invention into an expression vector and transducing the vector into a cell for expression.

[0024]

Fab' is an antibody fragment having a molecular weight of approximately 50,000 with antigen binding activity and cleaved SS bonds between F(ab') ₂ hinges. The Fab' used in the present invention can be obtained by treating the F(ab') _{2 of} the monoclonal antibody of the present invention with dithiothreitol as a reducing agent. The Fab' can also be obtained by inserting the DNA encoding the Fab' from the monoclonal antibody of the present invention into an expression vector for cells and transducing the vector into E. coli, yeast or animal cells for expression.

[0025]

F(ab') ₂ is an antibody fragment having a molecular weight of approximately 100,000 with antigen binding activity and formed by the binding of two Fab' regions in the hinge region obtained by breaking the bottom of the two SS bonds in the IgG hinge region with the enzyme pepsin. F(ab') ₂ used in the present invention can be obtained by treating the monoclonal antibody of the present invention with pepsin. Alternatively, the F(ab') ₂ used in the present invention can also be obtained by inserting the DNA encoding the F(ab') ₂ from a monoclonal antibody into an expression vector for cells and transducing the vector into E. coli, yeast, or animal cells for expression. .

[0026]

ScFv is a VH-P-VL or VL-P-VH polypeptide in which one VH chain and one VL chain are connected using an appropriate peptide linker (hereinafter referred to as P) and is an antibody fragment having antigenic activity. The VH and VL contained in the scFv used in the present invention may be derived from the monoclonal antibody of the present invention. The ScFv used in the present invention can be obtained by constructing an scFV expression vector using cDNA encoding VH and VL from the monoclonal antibody of the present invention, and transducing the vector into E. coli, yeast, or animal cells for expression.

[0027]

dsFv is produced by linking polypeptides in which each amino acid residue in VH and VL is replaced by a cysteine residue through an S-S bond. The amino acid residue replaced with a cysteine residue can be selected based on the prediction of the tertiary structure of the antibody according to the method described by Reiter et al. (Protein Engineering, 7, 697 (1994)). The VH or VL contained in the dsFv of the present invention can be obtained from the monoclonal antibody of the present invention. dsFv used in the present invention can be obtained by inserting into an appropriate expression vector using cDNA encoding VH and VL from the monoclonal antibody of the present invention to construct a dsFV expression vector, and by transducing the vector into E. coli, yeast, or animal cells to express .

[0028]

A diabody is an antibody fragment with scFv having the same or different antigen binding specificity forming a dimer and an antibody fragment with bivalent binding activity for the same antigen or with two types of antigen binding activities specific for different antigens. For example, a divalent diabody that specifically interacts with a monoclonal antibody of the present invention can be obtained by constructing a DNA encoding scFV with a peptide linker of 3 to 10 residues in length, using a cDNA encoding VH and VL of a monoclonal antibody of the present invention, inserting the DNA into a cellular expression vector for cells and transduction with the vector of E. coli, yeast or animal cells to express the diabody.

[0029]

The CDR-containing peptide includes at least one or more CDR regions in VH or VL. Many CDRs can be linked directly or via an appropriate peptide linker. The CDR-containing peptide of the present invention can be obtained by constructing a DNA encoding a CDR using a cDNA encoding VH and VL of a monoclonal antibody of the present invention, inserting the DNA into an expression vector for animal cells, and transducing the vector into E. coli, yeast, or animal cells. for expression. The CDR-containing peptide can also be prepared by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (tert-butyloxycarbonyl method).

[0030]

The monoclonal antibody or fragment thereof of the present invention is characterized by specific binding to Nav1.7. An example of how to measure the ability to specifically bind to Nav1.7 is shown below.

[0031]

Specific bonds may have an equilibrium dissociation constant of at least 1×10 ^-6 M or less (eg, lower KD represents stronger bonds). Methods for determining whether two molecules specifically bind are well known in the art and include the competitive ELISA method described in Example 5 and surface plasmon resonance. However, an isolated antibody that specifically binds to Nav1.7 may be cross-reactive with other antigens, such as Nav1.7 molecules derived from other species. However, a multispecific antibody that binds to hNav1.7 and one or more additional antigens, or a bispecific antibody that binds to two different regions of hNav1.7 (e.g., EC 3-1 loop and EC 3-3 loop), is considered an antibody that "specifically binds" to hNav1.7.

[0032]

The monoclonal antibody of the present invention, or a fragment thereof, is characterized by inhibition of Nav1.7. The following is an example of a method for measuring the ability to inhibit Nav1.7.

[0033]

The DNA encoding Nav1.7 was cloned into pcDNA3.1 (Invitrogen). The expression vector was transfected into FreeStyle 293 cells (Thermo Fisher Scientific) to construct cells with stable expression of Nav1.7. Specific inhibition of Nav1.7 by the antibody is assessed by performing manual voltage clamping using this cell according to the methods described below. Glass pieces coated with poly-L-lysine are placed in cups with a diameter of 35 mm. Cells with stable expression of Nav1.7 suspended in 10% FBS-containing DMEM (SIGMA) are plated on plates (4×10 ⁴ cells/dish). One day after inoculation, the glass pieces are transferred to a measuring chamber for the formation of whole cells. The antibody is treated with fixation -70 mV square wave 10 ms at 0.1 Hz. The measurement is carried out before and after antibody treatment (action takes >2 minutes).

[0034]

The monoclonal antibody of the present invention can be obtained using the CDRs or variable regions of the heavy and light chains described in the present description, in accordance with conventional methods in this field.

[0035]

The monoclonal antibody of the present invention further comprises a humanized monoclonal antibody. The humanized antibody has reduced antigenicity in the human body, so it is suitable for administration to humans for therapy and the like. A humanized monoclonal antibody is produced by grafting a complementarity determining region (CDR) of an antibody from a non-human mammal, such as a mouse, into the framework region (FR) of a human antibody. Thus, the FR region is derived from a human antibody. Suitable FRs can be selected by referring to the literature of Kabat E A. et al. The FR can be chosen such that the CDR can form the appropriate antigen binding site. If necessary, the amino acids of the FR variable regions can be changed so that the CDRs of the reconstituted humanized antibody can form the appropriate antigen binding site (Sato, K. et al., Cancer Res. Vol. 53, p. 851, 1993). The proportion of substituted amino acids in the FR is 0 to 15% of the total FR region, preferably 0 to 5% of the total FR region.

[0036]

In addition, a human antibody constant region is available as a humanized antibody of the present invention. As a preferred human antibody constant region, Cγ can be used for the heavy chain, for example, Cγ1, Cγ2, Cγ3 and Cγ4 can be used. For a light chain, Cκ and Cλ can be used. In addition, the C region of a human antibody can be modified to improve the stability of the antibody or its productivity. When humanized, the available human antibody can be of any isotype, such as IgG, IgM, IgA, IgE, and IgD. In the present invention, IgG is preferred and IgG1 or IgG4 is most preferred.

[0037]

The humanized monoclonal antibody of the present invention may or may not have a lysine added to the C-terminus of the heavy chain constant regions. Preferably, it contains a light chain constant region from the amino acid sequence of SEQ ID NO: 64 and a heavy chain constant region from the amino acid sequence of SEQ ID NO: 62 or 63, and may or may not have a lysine added to the C-terminus of SEQ ID NO: 62 or 63.

[0038]

Humanized monoclonal antibody can be obtained by conventional production methods (see, for example, Example 4 below, publication WO95/14041 and publication WO96/02576). In particular, a DNA sequence encoding a variable region designed to bind a mouse antibody CDR to a human antibody FR is synthesized by PCR from a series of oligonucleotides prepared with partially overlapping ends (see WO98/13388). The resulting DNA is ligated with DNA encoding a human antibody constant region and then inserted into an expression vector. Alternatively, the DNA encoding the antibody variable region can be inserted into an expression vector containing the antibody constant region DNA. For the production of antibodies of the present invention, antibody genes are inserted into expression vectors for expression under the control of expression control regions, eg, enhancers/promoters. The host cells can then be transformed with the expression vector to express the antibody.

[0039]

Host cells of the above transformants include vertebrate cells such as COS cells or CHO cells, prokaryotes and yeasts. Transformants can be cultured by methods known to the skilled person. Through cultivation, the monoclonal antibody of the present invention is produced inside or outside the transformants. The culture medium can be appropriately selected from conventional media depending on the host cell. In the case of COS cells, RPMI-1640 medium and Dulbecco's Modified Eagle's Minimal Essential Medium (DMEM) are available, and serum ingredients can be added as needed, as well as fetal bovine serum (FBS). The temperature for growing transformants is not limited as long as it does not seriously reduce the ability to produce proteins in the cell. Temperatures between 32 and 42ºC are preferred. The most preferred temperature is 37ºC. As necessary, cultivation can be carried out in an atmosphere containing carbon dioxide from 1 to 10% (v/v).

[0040]

Fractions containing a monoclonal antibody of the present invention, which are obtained in transformants or outside of transformants by the previously described methods, can be separated and purified by previously known separation methods. These methods are based on a physical or chemical property of the target protein. Specifically, examples include treatment with a conventional protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, various chromatography such as high performance liquid chromatography (HPLC), dialysis, and combinations thereof. The methods allow the monoclonal antibody of the present invention to be easily produced in high yield and purity.

[0041]

The monoclonal antibody or active fragment thereof of the present invention can be further modified with various molecules such as polyethylene glycols (PEGs), radioactive substances, toxins, and the like. Methods for modifying antibodies are known in the art.

[0042]

The monoclonal antibody of the present invention can also be fused to another protein at their N- or C-termini (Clinical Cancer Research, 2004, 10, 1274-1281). The artisan can roughly select the fusion protein.

[0043]

A pharmaceutical composition containing a monoclonal antibody of the present invention or a fragment thereof (pharmaceutical composition of the present invention) can be administered systemically or topically orally or parenterally. For parenteral administration, for example, intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, intranasal administration, inhalation and the like can be selected.

[0044]

The pharmaceutical composition of the present invention is very useful as a drug for the treatment and/or prevention of diseases associated with Nav1.7.

Diseases associated with Nav1.7 include pain, itching, neurogenic inflammation, cough, and the like.

"Pain" includes acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis, migraine, cluster headaches, trigeminal neuralgia, herpetic neuralgia, generalized neuralgia, neurodegenerative diseases, movement disorders, neuroendocrine pain, ataxia, sepsis , visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, sciatica, spinal pain, pain in the head or neck, severe pain or intractable pain, sudden pain, pain after surgery, genetic erythromelalgia, toothache, rhinitis, cancer pain, bladder disorders.

"Itching" includes acute pruritus, chronic pruritus, histamine-dependent pruritus, histamine-independent pruritus.

"Neurogenic inflammation" may be associated with asthma, arthritis, eczema, headache, migraine, psoriasis, or a combination.

"Cough" includes pathological or chronic cough.

[0045]

An effective dose is selected in the range from 0.01 mg to 100 mg per 1 kg of body weight at a time. Alternatively, a dose of 5 to 5000 mg can be selected, preferably a dose of 10 to 500 mg per patient. However, the dose of the pharmaceutical composition containing the monoclonal antibody of the present invention or a fragment thereof is not limited to these doses. The duration of administration may also be appropriately selected depending on age, symptom, and the like. at the patient. The pharmaceutical composition of the present invention may also include a pharmaceutically acceptable carrier or additive, depending on the route of administration. Examples of such a carrier and additive include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, water-soluble dextran, pectin, methylcellulose, ethylcellulose, casein, diglycerin, propylene glycol, polyethylene glycol, petrolatum, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants allowed as a pharmaceutical additive. The additive for use is appropriately selected or combined from the above depending on the dosage form, but is not limited to this.

[0046]

The present invention includes a polynucleotide encoding the heavy and/or light chain variable regions of a monoclonal antibody of the present invention. A polynucleotide encoding heavy chain variable regions of a monoclonal antibody of the present invention may further encode heavy chain constant regions. A polynucleotide encoding light chain variable regions of a monoclonal antibody of the present invention may further encode light chain constant regions. The present invention also relates to expression vectors containing at least one of these polynucleotides.

[0047]

A polynucleotide is a polymer composed of nucleotides such as several deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) as it encodes an antibody of the present invention. They may include bases other than natural products. The polynucleotide of the present invention is available for production of antibodies by genetic engineering and is useful as a screening probe for antibodies having equivalent activity to an antibody of the present invention. That is, by using a polynucleotide encoding an antibody of the present invention, or a portion thereof, as a probe, and using a method such as a hybridization or gene amplification method such as PCR, DNA can be obtained that can hybridize to said polynucleotide under stringent conditions and encode an antibody. having equivalent activity with the antibody of the present invention. Such DNA is also included in the polynucleotide of the present invention.

[0048]

The hybridization method (Sambrook, J et al., Molecular cloning, 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) is well known to those skilled in the art. The condition for hybridization, for example, includes conditions of low stringency. Low stringency conditions mean that the wash step after hybridization is carried out, for example, at 0.1×SSC containing 0.1% SDS at 42°C, preferably at 0.1×SSC containing 0.1% SDS at 50° C. A more preferred hybridization condition is one with high stringency. High stringency condition means, for example, at 5xSSC containing 0.1% SDS at 65ºC. Under these conditions, at a higher temperature, efficient production of a polynucleotide with higher similarity is expected. With regard to stringency of hybridization, several factors are listed, such as temperature or salt concentration. The person skilled in the art can select these factors appropriately and can obtain similar stiffness.

[0049]

Antibodies functionally equivalent to the monoclonal antibody of the present invention have generally high similarity in amino acid sequence. These antibodies are encoded by a polynucleotide, which is obtained using the methods of hybridization or gene amplification described above. Included in the present invention are antibodies that are functionally equivalent to the antibodies of the present invention and have high similarity in the amino acid sequence of the antibodies. High similarity means at least more than 75% amino acid sequence similarity, preferably 85% similarity, and more preferably 95% similarity. An algorithm described in the literature is available for determining the similarity of polypeptides (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730).

[0050]

The monoclonal antibody of the present invention, or a fragment thereof, specifically binds to Nav1.7 and can therefore be used to detect Nav1.7 in biological samples. Biological samples include blood, plasma, serum, urine, organs, tissues, bone marrow, lymph nodes, and the like. Thus, a kit containing the monoclonal antibody of the present invention can be used as a Nav1.7 detection kit. This kit includes a monoclonal antibody of the present invention or a fragment thereof, and may also include a labeled secondary antibody, a substrate necessary for the detection of labels, a carrier, a wash buffer, a sample diluent, a substrate for an enzyme, a solution that stops the reaction, Nav1.7 protein in as a purified standard for the substrate, instructions for use, etc.

[Example]

[0051]

Hereinafter, the present invention will be specifically described with reference to Examples. However, the present invention is not limited to the examples below.

[0052]

Example 1: Obtaining antibodies to Nav1.7

The peptide (1418-SVNVDKQPKYEYSL (SEQ ID NO: 1)-1431; hCE3C peptides) corresponding to the C-terminal regions of the extracellular loop of the C domain and E3 of human Nav1.7 (UniProtKB/Swiss-Prot: Q15858) was chosen as the antigen.

A peptide (Cys-SVNVDKQPKYEYSL (SEQ ID NO: 1); Cys-hCE3C) was synthesized to which a Cys residue was added at the N-terminus (manufactured by Toray Corporation). The peptide was coupled to maleimidated giant clam hemocyanin (KLH, manufactured by Thermo Scientific Corporation) to produce an immunogen. Female A/J Jms Slc mice were immunized with this peptide-KLH complex using complete Freund's adjuvant. Then additional immunization with incomplete Freund's adjuvant was carried out four times.

Three days after the last immunization, the spleen was excised. Spleen cells and a mouse myeloma cell (p3x6363-Ag8., Tokyo-based Mass Laboratory) were fused using the PEG method and selected in a medium containing hypoxanthine, aminopterin and thymidine. Hybridoma producing bound antibodies were identified by ELISA methods using immunogenic peptides with culture supernatant.

[0053]

Example 2: Determination of antibody sequences

The amino acid sequences of the variable regions of the heavy and light chains of the antibody were determined from the hybridoma cells of established clones by conventional methods (Table 1).

[Table 1] mAb Variable regions SEQ ID SEQ ID Variable regions SEQ ID SEQ ID 3B2 heavy chain 2 CDR1 3 light chain 33 CDR1 34 CDR2 four CDR2 35 CDR3 5 CDR3 36 5E12 heavy chain 6 CDR1 7 light chain 37 CDR1 38 CDR2 eight CDR2 39 CDR3 9 CDR3 40 7B9 heavy chain ten CDR1 eleven light chain 41 CDR1 38 CDR2 12 CDR2 42 CDR3 13 CDR3 40 15C8 heavy chain fourteen CDR1 fifteen light chain 43 CDR1 34 CDR2 16 CDR2 35 CDR3 17 CDR3 36 15H6 heavy chain eighteen CDR1 fifteen light chain 44 CDR1 34 CDR2 19 CDR2 45 CDR3 17 CDR3 36 28B5 heavy chain twenty CDR1 21 light chain 46 CDR1 34 CDR2 22 CDR2 35 CDR3 17 CDR3 47 29G3 heavy chain 23 CDR1 fifteen light chain 48 CDR1 34 CDR2 24 CDR2 35 CDR3 25 CDR3 36 12H4 heavy chain 26 CDR1 27 light chain 49 CDR1 34 CDR2 28 CDR2 35 CDR3 29 CDR3 fifty 22D3 heavy chain thirty CDR1 31 light chain 51 CDR1 34 CDR2 32 CDR2 35 CDR3 29 CDR3 fifty 3B2/28B5 heavy chain 2 CDR1 3 light chain 46 CDR1 34 CDR2 four CDR2 35 CDR3 5 CDR3 47 3B2/15C8 heavy chain 2 CDR1 3 light chain 43 CDR1 34 CDR2 four CDR2 35 CDR3 5 CDR3 36 28B5/15C8 heavy chain twenty CDR1 21 light chain 43 CDR1 34 CDR2 22 CDR2 35 CDR3 17 CDR3 36

[0054] Example 3: Alignment of antibody sequences

The heavy and light chain amino acid sequences of 15C8, 15H6, 28B5 and 29G3 were Kabat numbered using abYsis antibody sequence analysis software and aligned with GENETIX gene analysis software (FIGS. 1 and 2). As a result, the amino acid sequences of 15C8, 15H6, 28B5 and 29G3 were similar, as described below.

The light chain CDR1 consisted of 16 amino acids from SEQ ID NO:34.

The light chain CDR2 consisted of seven amino acids K-V-S-N-R-Xaa1-S, where Xaa1 was F or I: SEQ ID NO:54.

The light chain CDR3 consisted of 9 amino acids S-Q-S-Xaa2-H-V-P-Xaa3-T, where Xaa2 was T or I and Xaa3 was F or W: SEQ ID NO: 55.

The heavy chain CDR1 consisted of 5 amino acids G-Y-Y-Xaa4-H, where Xaa4 was M or I: SEQ ID NO: 56.

The heavy chain CDR2 consisted of 17 amino acids L-I-I-P-Y-Xaa5-G-Xaa6-Xaa7-F-Y-N-Xaa8-K-F-Xaa9-G, where Xaa5 was S or N, Xaa6 was D or E, Xaa7 was T or I, Xaa8 was Q or P, and Xaa9 was K or R: SEQ ID NO:57.

The heavy chain CDR3 consisted of 9 amino acids A-Xaa10-V-S-Y-A-M-D-Y, where Xaa10 was E or D: SEQ ID NO:58.

[0055]

Example 4 Humanization of an Antibody

12H4 was humanized in the following way. Using the Absis sequence analysis software, human germline acceptor sequences similar to the V-gene sequences for the heavy and light chain domains in the mouse antibody amino acid sequences were searched for and selected. For J chain regions, IMGT (http://www.imgt.org/) was searched for sequences with high homology to mouse antibody DNA to obtain a human backbone sequence. The humanized antibody sequence shown in Table 2 was designed by implanting CDR1, CDR2, and CDR3 into the heavy and light chains of a mouse antibody to this human framework sequence, as determined by Kabat numbering (Wu, TT and Kabat, EA, J. Exp. Med Aug1;132(2):211-50 (1970)). h12H4, h12H4-2 and h12H4-3, hIgG4Pro (SEQ ID NO: 62) or hIgG1 (SEQ ID NO: 63) were used as the constant region for the heavy chain, and hIgK (SEQ ID NO: 64) was used for the light chain.

Humanized 12H4 (h12H4), h12H4-2 and h12H4-3 implanted with CDRs to other human germline acceptor sequences showed similar affinity for mouse antibody against immunogenic peptide (Cys-CE3C) as described in Example 5.

[Table 2] mAb Variable regions SEQ ID SEQ ID Variable regions SEQ ID SEQ ID h12H4 heavy chain 52 CDR1 27 heavy chain 53 CDR1 34 CDR2 28 CDR2 35 CDR3 29 CDR3 fifty h12H4-2 heavy chain 59 CDR1 27 heavy chain 53 CDR1 34 CDR2 28 CDR2 35 CDR3 29 CDR3 fifty h12H4-3 heavy chain 60 CDR1 27 heavy chain 61 CDR1 34 CDR2 28 CDR2 35 CDR3 29 CDR3 fifty

[0056]

Example 5: Affinity Score

Affinity for immunogenic peptides (Cys-hCE3C) was determined by the competitive ELISA method described below.

The diluted solution of the resulting mouse antibody was added to the immobilized anti-mouse IgG antibody plate and allowed to bind at room temperature for 3 hours. After washing three times with ELISA clearing buffer, biotin labeled immunogenic peptide and streptavidin-HRP (derived from PIERCE) were added. Simultaneously, a series of dilutions of unlabeled immunogenic peptide was added and the reaction was carried out overnight at 4ºC. After washing three times with the ELISA wash, TMB-Substrate Chromogen (manufactured by Thermo Fisher Scientific) was added for staining. The reaction was quenched with an equal volume of 0.05N sulfuric acid and the absorbance was measured at 450 nm. The concentration of unlabeled peptide that attenuated the biotin-labeled peptide signal by half was taken as the IC ₅₀ . As a result, the antibody showed strong affinity for the immune peptide as shown in Table 3.

[0057]

[Table 3] mAb IC ₅₀ (nM) 3B2 9.5 15C8 6.3 28B5 0.2 29G3 7.0 12H4 0.3 h12H4 0.4 h12H4-2 0.5 h12H4-3 0.5

[0058]

Example 6 Identification of Amino Acids Critical for Humanized 12H4 Binding

Humanized 12H4 (h12H4) was Kabat numbered and CDR determined using abYsis antibody sequencing software. The results are shown in FIG. 3A, 3B. In addition, a mutant was obtained in which a point mutation was introduced at the amino acid corresponding to the CDR, and the affinity was calculated by the method described in Example 5. At this time, the peptide (SVNVDKQPKYEYSL; SEQ ID NO: 1) except for the N-terminal Cys of the immunogenic peptide was used as the unlabeled peptide and the affinity for that peptide was calculated. The results for the light chain are shown in Table 4 and the results for the heavy chain are shown in Table 5. As a result, the Q90 and V94 light chain lost their affinity for this peptide when these positions were changed to another amino acid. Table 4 n.d. means undetectable. This suggests that light chain Q90 and V94 are particularly important amino acids for binding. At the same time, the affinity of the N28 light chain and the M99 heavy chain decreased more than 10-fold when this amino acid was replaced by another amino acid. Based on this fact, light chain N28 and heavy chain M99 are considered important amino acids for binding.

[0059]

[Table 4] Mutant IC ₅₀ ratio
(mutant/wt) CDR1 R24K 0.8 S25T 0.8 S26T 1.3 Q27N 1.5 S27aT 1.3 L27bI 1.8 V27cL 1.4 H27dR 2.1 S27eT 1.0 N28Q 10.0 G29A 0.8 N30Q 1.0 T31S 1.0 Y32F 3.9 L33I 0.9 H34R 1.3 CDR2 K50R 1.5 V51L 1.6 S52T 1.3 N53Q 1.4 R54K 1.4 F55L 2.3 S56T 1.5 CDR3 S89T 1.4 Q90N n.d. S91T 2.3 T92S 1.3 H93R 2.4 V94L n.d. P95Gz 1.5 W96Y 2.4 T97S 1.4

[0060]

[Table 5] Mutant IC ₅₀ ratio
(mutant/wt) CDR1 Y31F 0.6 Y32F 0.8 Y33F 1.0 I34L 0.9 Q35N 1.3 CDR2 W50Y 2.0 I51L 1.7 Y52aF 1.4 P52bG 1.0 G53A 1.1 N54Q 1.2 G55A 1.3 N56Q 1.3 S57T 1.2 N58Q 1.2 I59L 0.9 T60S 2.6 E61D 1.9 K62R 1.8 F63L 1.1 K64R 2.2 G65A 2.5 CDR3 I95L 1.2 F96L 2.3 T97S 2.1 T98S 1.6 M99L 32.0 V100L 0.6 G100aA 1.2 D101E 1.4 Y102F 0.4

[0061]

Example 7 Evaluation of drug efficacy

(7-1) Derivation of models of partial ligation of the sciatic nerve of the rat

The rat was anesthetized with isoflurane and the left leg was shaved. The skin of the upper thigh was incised and the muscles cut to expose the sciatic nerve. Approximately half of the sciatic nerve was tightly tied with nylon thread and the muscles and skin were sutured. It was considered a surgical operation. Similar actions, with the exception of ligation of the sciatic nerve, were performed on the right leg, as a simulation of the operation.

[0062]

(7-2) Evaluation of drug efficacy by topical intraplantar administration

The rat prepared in (7-1) was used to evaluate pain behavior in mechanical hyperalgesia with an analgesiometer (Randal Selitto method) two weeks after surgery. The hind limb of the rat was compressed such that the pressure stimulus was increased by 16 g per second on the analgesiometer, and the pressure when the rat showed an avoidance response was set as the pain threshold. The pain threshold was assessed for the left and right hind limbs and was considered the pain threshold before treatment. The selected animals had a pain threshold of 60-90 g during surgery and 100-175 g during sham surgery. For training animals, similar procedures were performed before measuring the pain threshold before treatment. The antibody of the present invention was obtained at a concentration of 300 μg/40 μl using PBS and injected 40 μl intraplantarly. Five minutes after administration, the pain threshold of the right and left hind limbs was assessed and considered the pain threshold after treatment. % Reciprocal values were calculated by the methods described below to assess the pain effects of the antibody.

To evaluate the effect of the antibody on pain, % symptom relief was calculated using the methods described below.

% relief of symptoms = (pain threshold after treatment on the operated paw - pain threshold before treatment on the operated paw) / (pain threshold after treatment in sham surgery - pain threshold before treatment in sham surgery) × 100

The results of 3B2, 15C8, 28B5, 29G3 and 12H4 are shown in FIG. 4. The antibody of the present invention inhibits Nav1.7 by intraplant administration and shows significant efficacy for pain behavior.

[0063]

(7-3) Evaluation of drug efficacy by intravenous administration

The rat prepared in (7-1) was used to assess the behavior of mechanical allodynia with a von Frey thread two weeks after surgery. The rat was placed in a plastic cage on wire mesh for acclimatization. After the introduction of the antibody, von Frey threads (0.4-26 g) were pressed against the paw of the rat from the side of the wire mesh. The rat begins to show an avoidance reaction at a certain value of thread pressure. This value was set as the pain threshold. The threshold was evaluated for the left and right hind legs and set as the pain threshold before treatment. The selected animals had a pain threshold of 0.6-2 g for surgical intervention and 8-15 g for simulated surgery. For training animals, similar procedures were performed before measuring the pain threshold before treatment. Received the antibody of the present invention at a concentration of 50 mg/kg using saline and administered intravenously. 0.5-72 hours after administration, the pain threshold of the right and left hind legs was measured. It was set as pain threshold after treatment.

To evaluate the effect of a compound on pain, % symptom relief was calculated using the methods described below.

% symptom relief = (log of pain threshold after treatment on the operated paw - log of pain threshold before treatment on the operated paw) / (log of pain threshold after treatment in sham surgery - log of pain threshold before treatment in sham surgery)

The result for humanized 12H4 (h12H4) is shown in FIG. 5. The antibody of the present invention showed significant drug efficacy in pain behavior when administered intravenously.

[Industrial applicability]

[0064]

The monoclonal antibody of the present invention, or a fragment thereof, can be used to detect Nav1.7 in biological samples. In addition, the pharmaceutical composition containing the monoclonal antibody of the present invention or a fragment thereof is very useful as a drug for the treatment or prevention of diseases associated with Nav1.7 related diseases.

--->

SEQUENCE LIST

<110> SHIONOGI & CO., LTD.

<120> Monoclonal antibodies to Nav1.7

<130> 19P00036WO

<160> 64

<170>PatentIn version 3.5

<210> 1

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> hCE3C peptide

<400> 1

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

1 5 10

<210> 2

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy Chain 3B2

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Leu Trp Asp Pro Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 3

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 3B2 VH

<400> 3

Trp Tyr Val Met Ser

fifteen

<210> 4

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 3B2 VH

<400> 4

Thr Ile Ser Ser Gly Gly Val Ser Thr Tyr Tyr Pro Asp Thr Met Lys

1 5 10 15

gly

<210> 5

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 3B2 VH

<400> 5

Leu Trp Asp Pro Tyr Ala Met Asp Tyr

fifteen

<210> 6

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain 5E12

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Val Tyr Asp Gly Asn Tyr Val Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 7

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 5E12 VH

<400> 7

Asp Thr Tyr Met His

fifteen

<210> 8

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 5E12 VH

<400> 8

Arg Ile Asp Pro Ala Asn Gly Asn Thr Ile Tyr Asp Ser Lys Phe Gln

1 5 10 15

gly

<210> 9

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 5E12 VH

<400> 9

Val Tyr Asp Gly Asn Tyr Val Gly Ala Met Asp Tyr

1 5 10

<210> 10

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy Chain 7B9

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Val Asp Asp Gly Asn Tyr Val Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 11

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 7B9 VH

<400> 11

Asp Thr Tyr Ile His

fifteen

<210> 12

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 7B9 VH

<400> 12

Arg Ile Asp Pro Ala Asn Gly Asn Thr Ile Tyr Ala Ser Lys Phe Gln

1 5 10 15

gly

<210> 13

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 7B9 VH

<400> 13

Val Asp Asp Gly Asn Tyr Val Gly Ala Met Asp Tyr

1 5 10

<210> 14

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain 15C8

<400> 14

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Leu Ile Ile Pro Tyr Ser Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Ile Gly Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Glu Val Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 15

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 15C8 VH, 15H6 VH and 29G3 VH

<400> 15

Gly Tyr Tyr Met His

fifteen

<210> 16

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 15C8 VH

<400> 16

Leu Ile Ile Pro Tyr Ser Gly Asp Thr Phe Tyr Asn Gln Lys Phe Lys

1 5 10 15

gly

<210> 17

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 15C8VH, 15H6VH and 28B5VH

<400> 17

Ala Glu Val Ser Tyr Ala Met Asp Tyr

fifteen

<210> 18

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain 15H6

<400> 18

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Leu Ile Ile Pro Tyr Ser Gly Glu Ile Phe Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Ile Gly Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Glu Val Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 19

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 15H6 VH

<400> 19

Leu Ile Ile Pro Tyr Ser Gly Glu Ile Phe Tyr Asn Gln Lys Phe Lys

1 5 10 15

gly

<210> 20

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy Chain 28B5

<400> 20

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Leu Ile Ile Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Pro Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Gly Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Glu Val Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 21

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 28B5 VH

<400> 21

Gly Tyr Tyr Ile His

fifteen

<210> 22

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 28B5 VH

<400> 22

Leu Ile Ile Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Pro Lys Phe Lys

1 5 10 15

gly

<210> 23

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy Chain 29G3

<400> 23

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

1 5 10 15

Ser Val Asn Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Leu Ile Ile Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Ala Leu Gly Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Val Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 24

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 29G3 VH

<400> 24

Leu Ile Ile Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe Arg

1 5 10 15

gly

<210> 25

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 29G3 VH

<400> 25

Ala Asp Val Ser Tyr Ala Met Asp Tyr

fifteen

<210> 26

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain 12H4

<400> 26

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

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Tyr

20 25 30

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

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Gly Asn Ser Asn Ile Thr Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 27

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 12H4 VH, h12H4 VH, h12H4-2 VH and h12H4-3 VH

<400> 27

Tyr Tyr Tyr Ile Gln

fifteen

<210> 28

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 12H4 VH, h12H4 VH, h12H4-2 VH and h12H4-3 VH

<400> 28

Trp Ile Tyr Pro Gly Asn Gly Asn Ser Asn Ile Thr Glu Lys Phe Lys

1 5 10 15

gly

<210> 29

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 12H4 VH, 22D3 VH, h12H4 VH, h12H4-2 VH and h12H4-3 VH

<400> 29

Ile Phe Thr Thr Met Val Gly Asp Tyr

fifteen

<210> 30

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy Chain 22D3

<400> 30

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

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr His Tyr

20 25 30

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

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Gly Asn Thr Asn Asn Thr Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 31

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 22D3 VH

<400> 31

His Tyr Tyr Ile Gln

fifteen

<210> 32

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 22D3 VH

<400> 32

Trp Ile Tyr Pro Gly Asn Gly Asn Thr Asn Asn Thr Glu Lys Phe Lys

1 5 10 15

gly

<210> 33

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 3B2

<400> 33

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser 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 Ile Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 34

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 3B2 VK, 15C8 VK, 15H6 VK, 28B5 VK, 29G3 VK, 12H4 VK, 22D3

VK, h12H4 VK, h12H4-2 VK and h12H4-3 VK

<400> 34

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 35

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 3B2 VK, 15C8 VK, 28B5 VK, 29G3 VK, 12H4 VK, 22D3VK, h12H4

VK, h12H4-2 VK and h12H4-3 VK

<400> 35

Lys Val Ser Asn Arg Phe Ser

fifteen

<210> 36

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 3B2 VK, 15C8 VK, 15H6 VK and 29G3 VK

<400> 36

Ser Gln Ser Thr His Val Pro Phe Thr

fifteen

<210> 37

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 5E12

<400> 37

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

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

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

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gly Phe Thr Leu Asn Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Tyr Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Arg Ile Lys Arg

100 105

<210> 38

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 5E12 VK and 7B9 VK

<400> 38

Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His

1 5 10

<210> 39

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 5E12 VK

<400> 39

Tyr Ala Ser Gln Ser Ile Ser

fifteen

<210> 40

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 5E12 VK and 7B9 VK

<400> 40

Gln Gln Ser Asn Tyr Trp Pro Phe Thr

fifteen

<210> 41

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 7B9 VK

<400> 41

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

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

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

35 40 45

Lys Tyr Thr Ser Gln 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 Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Tyr Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Gly Ile Lys Arg

100 105

<210> 42

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 7B9 VK

<400> 42

Tyr Thr Ser Gln Ser Ile Ser

fifteen

<210> 43

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 15C8

<400> 43

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser 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 Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 44

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 15H6

<400> 44

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Ile Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Lys Phe Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 45

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 15H6 VK

<400> 45

Lys Val Ser Asn Arg Ile Ser

fifteen

<210> 46

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 28B5

<400> 46

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser 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 Phe Cys Ser Gln Ser

85 90 95

Ile His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 47

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 28B5 VK

<400> 47

Ser Gln Ser Ile His Val Pro Trp Thr

fifteen

<210> 48

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 29G3

<400> 48

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

1 5 10 15

Asp Gln Ala Ser Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser 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 Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 49

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 12H4

<400> 49

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Thr Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 50

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 12H4 VK, h12H4 VK, h12H4-2 VK and h12H4-3 VK

<400> 50

Ser Gln Ser Thr His Val Pro Trp Thr

fifteen

<210> 51

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain 22D3

<400> 51

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser 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 Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 52

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain h12H4

<400> 52

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Tyr

20 25 30

Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Gly Asn Ser Asn Ile Thr Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 53

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain h12H4 and h12H4-2

<400> 53

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr

<210> 54

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 VK

<220>

<221> other attribute

<222>(6)..(6)

<223> Xaa can be Ile or Phe

<400> 54

Lys Val Ser Asn Arg Xaa Ser

fifteen

<210> 55

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 VK

<220>

<221> other sign

<222> (4)..(4)

<223> Xaa can be Thr or Ile

<220>

<221> other attribute

<222>(8)..(8)

<223> Xaa can be Phe or Trp

<400> 55

Ser Gln Ser Xaa His Val Pro Xaa Thr

fifteen

<210> 56

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1 VH

<220>

<221> other sign

<222> (4)..(4)

<223> Xaa can be Met or Ile

<400> 56

Gly Tyr Tyr Xaa His

fifteen

<210> 57

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 VH

<220>

<221> other sign

<222>(6)..(6)

<223> Xaa can be Ser or Asn

<220>

<221> other sign

<222>(8)..(8)

<223> Xaa can be Asp or Glu

<220>

<221> other attribute

<222>(9)..(9)

<223> Xaa can be Thr or Ile

<220>

<221> other sign

<222> (13)..(13)

<223> Xaa can be Gln or Pro

<220>

<221> other sign

<222> (16)..(16)

<223> Xaa can be Lys or Arg

<400> 57

Leu Ile Ile Pro Tyr Xaa Gly Xaa Xaa Phe Tyr Asn Xaa Lys Phe Xaa

1 5 10 15

gly

<210> 58

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 VH

<220>

<221> other sign

<222> (2)..(2)

<223> Xaa can be Glu or Asp

<400> 58

Ala Xaa Val Ser Tyr Ala Met Asp Tyr

fifteen

<210> 59

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain h12H4-2

<400> 59

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Tyr

20 25 30

Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Gly Asn Ser Asn Ile Thr Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 60

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain h12H4-3

<400> 60

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Tyr

20 25 30

Tyr Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Gly Asn Ser Asn Ile Thr Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 61

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain h12H4-3

<400> 61

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr

<210> 62

<211> 327

<212> Protein

<213> Artificial sequence

<220>

<223> hIgG4Pro

<400> 62

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 Leu 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 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> 63

<211> 330

<212> Protein

<213> Artificial sequence

<220>

<223>hIgG1

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 64

<211> 105

<212> Protein

<213> Artificial sequence

<220>

<223> hIgK

<400> 64

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu

1 5 10 15

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

20 25 30

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<---

Claims (101)

1. A monoclonal antibody or fragment thereof that specifically binds to Nav1.7, having heavy chain variable region containing a1-1) CDR1 with amino acid sequence SEQ ID NO:21, CDR2 with the amino acid sequence of SEQ ID NO:22 and CDR3 with the amino acid sequence of SEQ ID NO:17, or a1-2) CDR1 with the amino acid sequence of SEQ ID NO:3, CDR2 with the amino acid sequence of SEQ ID NO:4 and CDR3 with the amino acid sequence of SEQ ID NO:5, and light chain variable region containing CDR1 with amino acid sequence SEQ ID NO:34, CDR2 with the amino acid sequence of SEQ ID NO:35 and CDR3 with the amino acid sequence of SEQ ID NO:47; b1) a heavy chain variable region containing CDR1 with the amino acid sequence of SEQ ID NO:27, CDR2 with the amino acid sequence of SEQ ID NO:28 and CDR3 with the amino acid sequence of SEQ ID NO:29, and light chain variable region containing CDR1 with amino acid sequence SEQ ID NO:34, CDR2 with the amino acid sequence of SEQ ID NO:35 and CDR3 with the amino acid sequence of SEQ ID NO:50; heavy chain variable region containing c1-1) CDR1 with the amino acid sequence of SEQ ID NO:3, CDR2 with the amino acid sequence of SEQ ID NO:4 and CDR3 with the amino acid sequence SEQ ID NO:5, c1-2) CDR1 with amino acid sequence SEQ ID NO:15, CDR2 with the amino acid sequence of SEQ ID NO:16 and CDR3 with the amino acid sequence of SEQ ID NO:17, c1-3) CDR1 with amino acid sequence SEQ ID NO:15, CDR2 with the amino acid sequence of SEQ ID NO:24 and CDR3 with the amino acid sequence of SEQ ID NO:25, or c1-4) CDR1 with amino acid sequence SEQ ID NO:21, CDR2 with the amino acid sequence of SEQ ID NO:22 and CDR3 with the amino acid sequence of SEQ ID NO:17, and light chain variable region containing CDR1 with amino acid sequence SEQ ID NO:34, CDR2 with the amino acid sequence of SEQ ID NO:35 and CDR3 with the amino acid sequence of SEQ ID NO:36; d1) a heavy chain variable region containing CDR1 with amino acid sequence SEQ ID NO:7, CDR2 with the amino acid sequence of SEQ ID NO:8 and CDR3 with the amino acid sequence of SEQ ID NO:9, and light chain variable region containing CDR1 with amino acid sequence SEQ ID NO:38, CDR2 with the amino acid sequence of SEQ ID NO:39 and CDR3 with the amino acid sequence of SEQ ID NO:40; e1) heavy chain variable region containing CDR1 with the amino acid sequence of SEQ ID NO:11, CDR2 with the amino acid sequence of SEQ ID NO:12 and CDR3 with the amino acid sequence of SEQ ID NO:13, and light chain variable region containing CDR1 with amino acid sequence SEQ ID NO:38, CDR2 with the amino acid sequence of SEQ ID NO:42 and CDR3 with the amino acid sequence of SEQ ID NO:40; f1) a heavy chain variable region containing CDR1 with amino acid sequence SEQ ID NO:15, CDR2 with the amino acid sequence of SEQ ID NO:19 and CDR3 with the amino acid sequence of SEQ ID NO:17, and light chain variable region containing CDR1 with amino acid sequence SEQ ID NO:34, CDR2 with the amino acid sequence of SEQ ID NO:45 and CDR3 with the amino acid sequence of SEQ ID NO:36; g1) heavy chain variable region containing CDR1 with the amino acid sequence of SEQ ID NO:31, CDR2 with the amino acid sequence of SEQ ID NO:32 and CDR3 with the amino acid sequence of SEQ ID NO:29, and light chain variable region containing CDR1 with amino acid sequence SEQ ID NO:34, CDR2 with the amino acid sequence of SEQ ID NO:35 and CDR3 with the amino acid sequence of SEQ ID NO:50; h1) a heavy chain variable region containing CDR1 with amino acid sequence SEQ ID NO:56, CDR2 with the amino acid sequence of SEQ ID NO:57 and CDR3 with the amino acid sequence of SEQ ID NO:58, and light chain variable region containing CDR1 with amino acid sequence SEQ ID NO:34, CDR2 with the amino acid sequence of SEQ ID NO:54 and CDR3 with the amino acid sequence of SEQ ID NO:55. 2. An antibody or fragment according to claim 1, having a heavy chain variable region with the amino acid sequence of SEQ ID NO:52, 59, or 60, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:52, 59, or 60, and a light chain variable region with an amino acid sequence of SEQ ID NO:53 or 61, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:53 or 61. 3. An antibody or fragment according to claim 2, having a1-1) a heavy chain variable region with the amino acid sequence of SEQ ID NO:52, and the variable region of the light chain with the amino acid sequence of SEQ ID NO:53; a1-2) a heavy chain variable region with the amino acid sequence of SEQ ID NO:59, and the variable region of the light chain with the amino acid sequence of SEQ ID NO:53; or a1-3) a heavy chain variable region with the amino acid sequence of SEQ ID NO:60, and the variable region of the light chain with the amino acid sequence of SEQ ID NO:61. 4. An antibody or fragment thereof according to any one of claims 1 to 3, further having a heavy chain constant region with the amino acid sequence of SEQ ID NO:62 or 63, and a light chain constant region with the amino acid sequence of SEQ ID NO:64. 5. Pharmaceutical composition for the treatment or prevention of Nav1.7-related diseases, containing an effective dose of the antibody or fragment thereof according to any one of claims 1-4. 6. Pharmaceutical composition according to claim 5 for the treatment or prevention of pain and/or itching. 7. A polynucleotide encoding the heavy chain variable region of an antibody according to claim 2 or 3, where the polynucleotide optionally encodes the constant region of the heavy chain of the antibody according to claim 4. 8. A polynucleotide encoding a light chain variable region of an antibody according to claim 2 or 3, where the polynucleotide optionally encodes the constant region of the light chain of the antibody according to claim 4. 9. An expression vector containing a polynucleotide according to claim 7 or 8.

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