Binding molecules specific for LIF and uses thereof

Abstract

The present invention provides binding molecules specific for LIF and uses thereof, and in particular, provides an isolated antibody or antigen-binding portion thereof that binds to LIF and inhibits LIF activity. The present invention also provides uses of the isolated antibody or antigen-binding portion thereof of the present invention in treating diseases.

Description

Technical Field

[0001] The present invention relates to isolated antibodies or antigen-binding portions thereof that specifically bind to LIF, as well as uses of the isolated antibodies or antigen-binding portions thereof of the present invention, and therapeutic methods using the isolated antibodies or antigen-binding portions thereof of the present invention. Background of the Invention

[0003] Leukemia inhibitory factor (LIF) is a member of the IL-6 class of cytokines. It has multiple biological activities, including stimulating or inhibiting cell proliferation, differentiation, and survival [1]. Human LIF protein contains 202 amino acids. It has two receptors on the cell membrane surface, GP130 and LIFR. LIF protein binds to these two receptors, causing the two receptors to form heterodimers, thereby activating downstream signaling pathways, such as the MAPK signaling pathway and the JAK / STAT signaling pathway [2]. High expression and high serum concentrations of LIF protein have been shown to be associated with poor prognosis in a variety of tumors [3,4]. LIF is an important regulator of tumor stem cells, playing an important role in stem cell maintenance, self-renewal, and pluripotency, and is associated with chemotherapy resistance [5,6]. In addition, LIF can also promote tumor growth and metastasis [7]. Recent evidence shows that in tumors, LIF upregulates the JAK-STAT3 signaling pathway in an autocrine and paracrine manner, thereby promoting tumor growth and inhibiting immune responses [8,9,10]. Therefore, LIF is a highly promising therapeutic target, but the current treatment methods developed for LIF are not optimistic. For example, many literature reports have shown that using RNA interference to reduce the expression of LIF protein can inhibit tumor growth [11,12], but RNA interference technology has weaknesses such as poor targeting, short half-life, and poor membrane permeability, making it difficult to develop into a drug; EC359 is a small molecule inhibitor targeting LIFR, which can not only inhibit the binding of LIFR to LIF but also simultaneously inhibit the binding of OSM, CTF1, and CNTF to LIFR

[13] . Whether these additional inhibitions will lead to additional toxicity is still unknown, and small molecule inhibitors specific for LIF protein have not been reported; currently, only one antibody targeting LIF protein is in the clinical development stage, and the relevant safety and efficacy data have not been made public.

[0004] Therefore, more research is still needed to develop drugs and combination treatments targeting LIF. Summary of the invention

[0005] The present invention provides isolated antibodies or antigen-binding portions that specifically bind to LIF and their use in treating disease.

[0006] In one aspect, the present invention provides an isolated antibody or an antigen-binding portion thereof, wherein the isolated antibody or the antigen-binding portion thereof binds to an epitope shown in the amino acid sequence TYGPDTSGKDVFQKK (SEQ ID NO: 61) of the human LIF protein or an epitope of the corresponding amino acid sequence in different mammalian species.

[0007] In yet another aspect, the present invention provides an isolated antibody or antigen-binding portion thereof, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0008] (a) LCDR1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 66 and conservative modifications thereof;

[0009] (b) LCDR2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 67 and conservative modifications thereof;

[0010] (c) LCDR3, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or SEQ ID NO: 68 and conservative modifications thereof;

[0011] (d) HCDR1, the HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or SEQ ID NO: 69 and conservative modifications thereof;

[0012] (e) HCDR2, the HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 45 or SEQ ID NO: 70 and conservative modifications thereof; and

[0013] (f) HCDR3, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 or SEQ ID NO: 71 and conservative modifications thereof.

[0014] Optionally, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 has additions, substitutions, deletions and / or insertions of 17 or fewer amino acids.

[0015] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0016] (a) LCDR1, wherein the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and conservatively modified forms thereof;

[0017] (b) LCDR2, which LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and conservatively modified forms thereof;

[0018] (c) LCDR3, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and conservatively modified forms thereof;

[0019] (d) HCDR1, wherein the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and conservatively modified forms thereof;

[0020] (e) HCDR2, the HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or SEQ ID NO: 45 and conservative modifications thereof; and

[0021] (f) HCDR3, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and conservative modifications thereof.

[0022] Optionally, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 has additions, substitutions, deletions and / or insertions of 17 or fewer amino acids.

[0023] In certain embodiments, optionally, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 has additions, substitutions, deletions, and / or insertions of 9 or fewer amino acids.

[0024] In certain embodiments, optionally, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 has additions, substitutions, deletions, and / or insertions of 5 or fewer amino acids.

[0025] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0026] 1) (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c)

[0027] (d) comprising a LCDR3 of SEQ ID NO: 3, (d) comprising a HCDR1 of SEQ ID NO: 4, (e) comprising a HCDR2 of SEQ ID NO: 5, and (f) comprising a HCDR3 of SEQ ID NO: 6;

[0028] 2) (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c)

[0029] (d) comprising a LCDR3 of SEQ ID NO: 3, (d) comprising a HCDR1 of SEQ ID NO: 4, (e) comprising a HCDR2 of SEQ ID NO: 45, and (f) comprising a HCDR3 of SEQ ID NO: 6; or

[0030] 3) (a) LCDR1 comprising SEQ ID NO: 66, (b) LCDR2 comprising SEQ ID NO: 67, (c)

[0031] The LCDR3 of SEQ ID NO: 68, (d) comprising the HCDR1 of SEQ ID NO: 69, (e) comprising the

[0032] NO:70, and (f) comprising HCDR3 of SEQ ID NO:71.

[0033] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0034] 1) (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c)

[0035] (d) comprising a LCDR3 of SEQ ID NO: 3, (d) comprising a HCDR1 of SEQ ID NO: 4, (e) comprising a HCDR2 of SEQ ID NO: 5, and (f) comprising a HCDR3 of SEQ ID NO: 6; or

[0036] 2) (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c)

[0037] (d) comprising a LCDR3 of SEQ ID NO:3, (d) comprising a HCDR1 of SEQ ID NO:4, (e) comprising a HCDR2 of SEQ ID NO:45, and (f) comprising a HCDR3 of SEQ ID NO:6.

[0038] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0039] (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c) LCDR3 comprising SEQ ID NO: 3, (d) HCDR1 comprising SEQ ID NO: 4, (e) HCDR2 comprising SEQ ID NO: 5, and (f) HCDR3 comprising SEQ ID NO: 6.

[0040] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0041] (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c) LCDR3 comprising SEQ ID NO: 3, (d) HCDR1 comprising SEQ ID NO: 4, (e) HCDR2 comprising SEQ ID NO: 45, and (f) HCDR3 comprising SEQ ID NO: 6.

[0042] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0043] (a) LCDR1 comprising SEQ ID NO:66, (b) LCDR2 comprising SEQ ID NO:67, (c) LCDR3 comprising SEQ ID NO:68, (d) HCDR1 comprising SEQ ID NO:69, (e) HCDR2 comprising SEQ ID NO:70, and (f) HCDR3 comprising SEQ ID NO:71.

[0044] In certain embodiments, the isolated antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

[0045] In certain embodiments, the isolated antibody is a humanized antibody comprising a framework region derived from a human antibody or a framework region variant thereof.

[0046] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0047] (i) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 11, 15, 19, 46, 74 or 82 and conservatively modified versions thereof; and

[0048] (ii) a heavy chain variable region (VH), which comprises an amino acid sequence that is at least 85% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 27, 31, 48, 75 or 83 and conservatively modified versions thereof.

[0049] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0050] (i) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 11, 15, 19 or 46 and conservatively modified versions thereof; and

[0051] (ii) a heavy chain variable region (VH), which comprises an amino acid sequence that is at least 85% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 27, 31 or 48 and conservatively modified versions thereof.

[0052] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0053] (i) a light chain variable region (VL), the light chain variable region comprising an amino acid sequence that is at least 85% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 11, 15 or 19 and conservatively modified versions thereof;

[0054] as well as

[0055] (ii) a heavy chain variable region (VH), comprising an amino acid sequence having at least 85% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 27 or 31 and conservatively modified versions thereof.

[0056] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the light chain variable region selected from (i); the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the heavy chain variable region selected from (ii).

[0057] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0058] 1) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 23;

[0059] 2) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 27;

[0060] 3) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 31;

[0061] 4) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 23;

[0062] 5) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 27;

[0063] 6) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 31;

[0064] 7) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 23;

[0065] 8) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 27;

[0066] 9) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 31;

[0067] 10) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 23;

[0068] 11) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 27;

[0069] 12) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 31;

[0070] 13) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 46; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 48;

[0071] 14) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 74; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 75; or

[0072] 15) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 82; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 83.

[0073] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0074] 1) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 23;

[0075] 2) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 27;

[0076] 3) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 31;

[0077] 4) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 23;

[0078] 5) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 27;

[0079] 6) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 31;

[0080] 7) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 23;

[0081] 8) a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 27;

[0082] 9) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 31;

[0083] 10) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 23;

[0084] 11) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 27; or

[0085] 12) a light chain variable region (VL) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 31.

[0086] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0087] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:7; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:23.

[0088] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0089] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:7; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:27.

[0090] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0091] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:7; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:31.

[0092] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0093] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 23.

[0094] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0095] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 27.

[0096] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0097] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 31.

[0098] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0099] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 23.

[0100] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0101] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 27.

[0102] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0103] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 31.

[0104] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0105] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 23.

[0106] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0107] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 27.

[0108] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0109] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 31.

[0110] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0111] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:46; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:48.

[0112] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0113] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:74; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:75.

[0114] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0115] a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:82; and a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:83.

[0116] In certain embodiments, the light chain variable region and heavy chain variable region respectively comprise amino acid sequences that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the light chain variable region and heavy chain variable region selected from 1)-15).

[0117] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region combination selected from the following (i) or (ii):

[0118] (i) a light chain variable region (VL), which comprises LCDR1, LCDR2 and LCDR3 having the same sequence as any one of SEQ ID NOs: 7, 11, 15, 19, 46 or 82; and

[0119] (ii) a heavy chain variable region (VH), which comprises HCDR1, HCDR2 and HCDR3 identical to any one of SEQ ID NOs: 23, 27, 31, 48 or 83.

[0120] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein:

[0121] (I) the light chain comprises an amino acid sequence having at least 85% homology to an amino acid sequence selected from the group consisting of:

[0122] SEQ ID NOs: 9, 13, 17, 21, 37, 39, 50 or 54 and conservatively modified amino acid sequences thereof;

[0123] as well as

[0124] (II) the heavy chain comprises an amino acid sequence having at least 85% homology to an amino acid sequence selected from the group consisting of:

[0125] The amino acid sequences of SEQ ID NOs: 25, 29, 33, 35, 52 or 56 and conservatively modified forms thereof.

[0126] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein:

[0127] (I) the light chain comprises an amino acid sequence having at least 85% homology to an amino acid sequence selected from the group consisting of:

[0128] The amino acid sequence of SEQ ID NOs: 9, 13, 17, 21, 37, 39 or 50 and conservative modifications thereof; and

[0129] (II) the heavy chain comprises an amino acid sequence having at least 85% homology to an amino acid sequence selected from the group consisting of:

[0130] The amino acid sequences of SEQ ID NOs: 25, 29, 33, 35 or 52 and conservatively modified forms thereof.

[0131] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein:

[0132] (I) the light chain comprises an amino acid sequence having at least 85% homology to an amino acid sequence selected from the group consisting of:

[0133] The amino acid sequence of SEQ ID NOs: 9, 13, 17 or 21 and conservative modifications thereof; and

[0134] (II) the heavy chain comprises an amino acid sequence having at least 85% homology to an amino acid sequence selected from the group consisting of:

[0135] The amino acid sequences of SEQ ID NOs: 25, 29 or 33 and conservatively modified forms thereof.

[0136] In certain embodiments, the light chain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the light chain selected from (I); the heavy chain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the heavy chain selected from (II).

[0137] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0138] 1) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 25;

[0139] 2) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 29;

[0140] 3) a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 33;

[0141] 4) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 13; and a heavy chain

[0142] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 25;

[0143] 5) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 13; and a heavy chain

[0144] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 29;

[0145] 6) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 13; and a heavy chain

[0146] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 33;

[0147] 7) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 17; and a heavy chain

[0148] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 25;

[0149] 8) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 17; and a heavy chain

[0150] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 29;

[0151] 9) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 17; and a heavy chain

[0152] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 33;

[0153] 10) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 21; and a heavy chain comprising:

[0154] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 25;

[0155] 11) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 21; and a heavy chain

[0156] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 29;

[0157] 12) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 21; and a heavy chain comprising:

[0158] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 33;

[0159] 13) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 37; and a heavy chain comprising:

[0160] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 35;

[0161] 14) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 39; and a heavy chain comprising:

[0162] a chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 35;

[0163] 15) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 50; and a heavy chain comprising:

[0164] A chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 52; or

[0165] 16) a light chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO: 54; and a heavy chain comprising:

[0166] A chain comprising an amino acid sequence that is at least 85% homologous to the amino acid sequence of SEQ ID NO:56.

[0167] In certain embodiments, the light chain and heavy chain respectively comprise an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the light chain and heavy chain selected from 1)-16).

[0168] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0169] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:9; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:25.

[0170] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0171] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:9; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:29.

[0172] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0173] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:9; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:33.

[0174] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0175] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 13; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 25.

[0176] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0177] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 13; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 29.

[0178] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0179] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 13; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 33.

[0180] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0181] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 17; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 25.

[0182] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0183] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 17; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 29.

[0184] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0185] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 17; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 33.

[0186] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0187] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:21; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:25.

[0188] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0189] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:21; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:29.

[0190] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0191] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:21; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:33.

[0192] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0193] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:37; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:35.

[0194] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0195] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:39; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:35.

[0196] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0197] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:50; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:52.

[0198] In certain embodiments, the isolated antibody or antigen-binding portion thereof comprises:

[0199] a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:54; and a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO:56.

[0200] In another aspect, the present invention provides an isolated antibody or antigen-binding portion thereof, wherein the isolated antibody or antigen-binding portion thereof comprises: (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c) LCDR3 comprising SEQ ID NO: 3, (d) HCDR1 comprising SEQ ID NO: 4, (e) HCDR2 comprising SEQ ID NO: 5, and (f) HCDR3 comprising SEQ ID NO: 6.

[0201] In another aspect, the present invention provides an isolated antibody or antigen-binding portion thereof, wherein the isolated antibody or antigen-binding portion thereof comprises: (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c) LCDR3 comprising SEQ ID NO: 3, (d) HCDR1 comprising SEQ ID NO: 4, (e) HCDR2 comprising SEQ ID NO: 45, and (f) HCDR3 comprising SEQ ID NO: 6.

[0202] In another aspect, the present invention provides an isolated antibody or antigen-binding portion thereof, comprising a light chain variable region (VL) as shown in SEQ ID NO: 7 and a heavy chain variable region (VH) as shown in SEQ ID NO: 23.

[0203] In yet another aspect, the present invention provides an isolated antibody or antigen-binding portion thereof, comprising a light chain variable region (VL) as shown in SEQ ID NO: 11 and a heavy chain variable region (VH) as shown in SEQ ID NO: 31.

[0204] In another aspect, the present invention provides an isolated antibody or antigen-binding portion thereof, comprising a light chain variable region (VL) as shown in SEQ ID NO: 19 and a heavy chain variable region (VH) as shown in SEQ ID NO: 31.

[0205] In certain embodiments, the isolated antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a bispecific antibody, Fv, a single chain antibody (scFv), Fab, Fab', Fab'-SH or F(ab')2.

[0206] In certain embodiments, the isolated antibody is IgG.

[0207] In certain embodiments, the isolated antibody is IgG1, IgG2, or IgG4.

[0208] In certain embodiments, the isolated antibody or antigen-binding portion thereof is a LIF (leukemia inhibitory factor) antagonist.

[0209] In certain embodiments, the isolated antibody or antigen-binding portion thereof is capable of inhibiting LIF expression and / or blocking LIF activity.

[0210] In certain embodiments, the isolated antibody or antigen-binding portion thereof is capable of competing or cross-competing for binding to LIF.

[0211] In yet another aspect, the present invention provides a nucleotide composition comprising a nucleotide molecule encoding an isolated antibody or antigen-binding portion thereof of the present invention. In some embodiments, the nucleotide molecule is DNA or RNA. In some embodiments, the nucleotide molecule is DNA.

[0212] In certain embodiments, the nucleotide composition comprises:

[0213] (i) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NOs: 7, 11, 15, 19, 46 or 82; and

[0214] (ii) a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NOs: 23, 27, 31, 48 or 83.

[0215] In certain embodiments, the nucleotide composition comprises:

[0216] (i) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NOs: 7, 11, 15, 19 or 46; and

[0217] (ii) a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NOs: 23, 27, 31 or 48.

[0218] In certain embodiments, the nucleotide composition comprises:

[0219] (i) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NOs: 7, 11, 15 or 19; and

[0220] (ii) a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NOs: 23, 27 or 31.

[0221] In certain embodiments, the first nucleic acid molecule comprises DNA that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the first nucleic acid molecule selected from (i); and the second nucleic acid molecule comprises DNA that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the second nucleic acid molecule selected from (ii).

[0222] In certain embodiments, the nucleotide composition comprises:

[0223] 1) A first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23

[0224] (VH) DNA;

[0225] 2) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 27

[0226] (VH) DNA;

[0227] 3) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 31

[0228] (VH) DNA;

[0229] 4) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23

[0230] (VH) DNA;

[0231] 5) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 27

[0232] (VH) DNA;

[0233] 6) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31.

[0234] DNA;

[0235] 7) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23;

[0236] 8) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 27

[0237] (VH) DNA;

[0238] 9) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31;

[0239] 10) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23;

[0240] 11) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27;

[0241] 12) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31;

[0242] 13) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 46; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 48;

[0243] 14) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 74; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 75; or

[0244] 15) A first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 82; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 83.

[0245] In certain embodiments, the nucleotide composition comprises:

[0246] 1) A first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23;

[0247] 2) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27;

[0248] 3) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31;

[0249] 4) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23

[0250] (VH) DNA;

[0251] 5) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27;

[0252] 6) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31.

[0253] DNA;

[0254] 7) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23;

[0255] 8) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 27

[0256] (VH) DNA;

[0257] 9) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31;

[0258] 10) a first nucleic acid sequence comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid sequence comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23;

[0259] 11) a first nucleic acid sequence comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid sequence comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27; or

[0260] 12) A first nucleic acid sequence comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid sequence comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31.

[0261] In certain embodiments, the nucleotide composition comprises:

[0262] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:7; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:23.

[0263] In certain embodiments, the nucleotide composition comprises:

[0264] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:7; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:27.

[0265] In certain embodiments, the nucleotide composition comprises:

[0266] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:7; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:31.

[0267] In certain embodiments, the nucleotide composition comprises:

[0268] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23.

[0269] In certain embodiments, the nucleotide composition comprises:

[0270] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27.

[0271] In certain embodiments, the nucleotide composition comprises:

[0272] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31.

[0273] In certain embodiments, the nucleotide composition comprises:

[0274] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23.

[0275] In certain embodiments, the nucleotide composition comprises:

[0276] The first nucleic acid molecule contains a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and the second nucleic acid molecule contains a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27.

[0277] In certain embodiments, the nucleotide composition comprises:

[0278] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31.

[0279] In certain embodiments, the nucleotide composition comprises:

[0280] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23.

[0281] In certain embodiments, the nucleotide composition comprises:

[0282] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27.

[0283] In certain embodiments, the nucleotide composition comprises:

[0284] The first nucleic acid molecule comprises a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and the second nucleic acid molecule comprises a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31.

[0285] In certain embodiments, the nucleotide composition comprises:

[0286] The first nucleic acid molecule contains a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:46; and the second nucleic acid molecule contains a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:48.

[0287] In certain embodiments, the nucleotide composition comprises:

[0288] The first nucleic acid molecule contains a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:74; and the second nucleic acid molecule contains a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:75.

[0289] In certain embodiments, the nucleotide composition comprises:

[0290] The first nucleic acid molecule contains a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:82; and the second nucleic acid molecule contains a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:83.

[0291] In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence respectively comprise DNA sequences that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the first nucleic acid sequence or the second nucleic acid sequence selected from 1)-15).

[0292] In certain embodiments, the DNA encoding the light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO:7 is set forth in SEQ ID NO:8.

[0293] In certain embodiments, the DNA encoding the light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO:11 is set forth in SEQ ID NO:12.

[0294] In certain embodiments, the DNA encoding the light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO:15 is set forth in SEQ ID NO:16.

[0295] In certain embodiments, the DNA encoding the light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO:19 is set forth in SEQ ID NO:20.

[0296] In certain embodiments, the DNA encoding the light chain variable region (VL) with the amino acid sequence set forth in SEQ ID NO:46 is set forth in SEQ ID NO:47.

[0297] In certain embodiments, the DNA encoding the light chain variable region (VL) with the amino acid sequence set forth in SEQ ID NO:74 is set forth in SEQ ID NO:76.

[0298] In certain embodiments, the DNA encoding the light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:82 is as shown in SEQ ID NO:72.

[0299] In certain embodiments, the DNA encoding the heavy chain variable region (VH) with the amino acid sequence set forth in SEQ ID NO:23 is set forth in SEQ ID NO:24.

[0300] In certain embodiments, the DNA encoding the heavy chain variable region (VH) with the amino acid sequence set forth in SEQ ID NO:27 is set forth in SEQ ID NO:28.

[0301] In certain embodiments, the DNA encoding the heavy chain variable region (VH) with the amino acid sequence set forth in SEQ ID NO:31 is set forth in SEQ ID NO:32.

[0302] In certain embodiments, the DNA encoding the heavy chain variable region (VH) with the amino acid sequence set forth in SEQ ID NO:48 is set forth in SEQ ID NO:49.

[0303] In certain embodiments, the DNA encoding the heavy chain variable region (VH) with the amino acid sequence set forth in SEQ ID NO:75 is set forth in SEQ ID NO:77.

[0304] In certain embodiments, the DNA encoding the heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO:83 is as shown in SEQ ID NO:73.

[0305] In certain embodiments, the nucleotide composition comprises:

[0306] (I) a first nucleic acid sequence comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NOs: 9, 13, 17, 21, 37, 39, 50 or 54; and

[0307] (II) A second nucleic acid sequence comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NOs: 25, 29, 33, 35, 52 or 56.

[0308] In certain embodiments, the nucleotide composition comprises:

[0309] (I) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NOs: 9, 13, 17, 21, 37, 39 or 50; and

[0310] (II) A second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NOs: 25, 29, 33, 35 or 52.

[0311] In certain embodiments, the nucleotide composition comprises:

[0312] (I) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NOs: 9, 13, 17 or 21; and

[0313] (II) A second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NOs: 25, 29 or 33.

[0314] In certain embodiments, the first nucleic acid molecule comprises DNA that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the first nucleic acid molecule selected from (I); the second nucleic acid molecule comprises DNA that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the second nucleic acid molecule selected from (II).

[0315] In certain embodiments, the nucleotide composition comprises:

[0316] 1) A first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 9; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25;

[0317] 2) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 9; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 29;

[0318] 3) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 9; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 33;

[0319] 4) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 13; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25;

[0320] 5) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 13; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 29;

[0321] 6) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 13; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 33;

[0322] 7) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25;

[0323] 8) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 29;

[0324] 9) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 33;

[0325] 10) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 21; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25;

[0326] 11) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 21; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 29;

[0327] 12) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 21; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 33;

[0328] 13) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 37; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 35;

[0329] 14) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 39; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 35;

[0330] 15) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 50; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 52; or

[0331] 16) A first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 54; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 56.

[0332] In certain embodiments, the first nucleic acid molecule and the second nucleic acid molecule comprise DNA that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the first nucleic acid molecule or the second nucleic acid molecule selected from 1)-16).

[0333] In certain embodiments, the nucleotide composition comprises:

[0334] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:9; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:25.

[0335] In certain embodiments, the nucleotide composition comprises:

[0336] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:9; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:29.

[0337] In certain embodiments, the nucleotide composition comprises:

[0338] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:9; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:33.

[0339] In certain embodiments, the nucleotide composition comprises:

[0340] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:13; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:25.

[0341] In certain embodiments, the nucleotide composition comprises:

[0342] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:13; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:29.

[0343] In certain embodiments, the nucleotide composition comprises:

[0344] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 13; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 33.

[0345] In certain embodiments, the nucleotide composition comprises:

[0346] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25.

[0347] In certain embodiments, the nucleotide composition comprises:

[0348] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 29.

[0349] In certain embodiments, the nucleotide composition comprises:

[0350] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 33.

[0351] In certain embodiments, the nucleotide composition comprises:

[0352] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:21; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:25.

[0353] In certain embodiments, the nucleotide composition comprises:

[0354] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:21; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:29.

[0355] In certain embodiments, the nucleotide composition comprises:

[0356] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:21; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:33.

[0357] In certain embodiments, the nucleotide composition comprises:

[0358] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:37; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:35.

[0359] In certain embodiments, the nucleotide composition comprises:

[0360] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:39; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:35.

[0361] In certain embodiments, the nucleotide composition comprises:

[0362] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:50; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:52.

[0363] In certain embodiments, the nucleotide composition comprises:

[0364] The first nucleic acid molecule comprises a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO:54; and the second nucleic acid molecule comprises a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO:56.

[0365] In certain embodiments, the DNA encoding the light chain having the amino acid sequence set forth in SEQ ID NO:9 is set forth in SEQ ID NO:10.

[0366] In certain embodiments, the DNA encoding the light chain having the amino acid sequence set forth in SEQ ID NO:13 is set forth in SEQ ID NO:14.

[0367] In certain embodiments, the DNA encoding the light chain having the amino acid sequence set forth in SEQ ID NO:17 is set forth in SEQ ID NO:18.

[0368] In certain embodiments, the DNA encoding the light chain having the amino acid sequence set forth in SEQ ID NO:21 is set forth in SEQ ID NO:22.

[0369] In certain embodiments, the DNA encoding the light chain having an amino acid sequence as shown in SEQ ID NO:37 is shown in SEQ ID NO:38.

[0370] In certain embodiments, the DNA encoding the light chain having the amino acid sequence set forth in SEQ ID NO:39 is set forth in SEQ ID NO:40.

[0371] In certain embodiments, the DNA encoding the light chain having the amino acid sequence shown in SEQ ID NO:50 is shown in SEQ ID NO:51.

[0372] In certain embodiments, the DNA encoding the light chain having an amino acid sequence as shown in SEQ ID NO:54 is as shown in SEQ ID NO:55.

[0373] In certain embodiments, the DNA encoding the heavy chain having the amino acid sequence set forth in SEQ ID NO:25 is set forth in SEQ ID NO:26.

[0374] In certain embodiments, the DNA encoding the heavy chain having the amino acid sequence set forth in SEQ ID NO:29 is set forth in SEQ ID NO:30.

[0375] In certain embodiments, the DNA encoding the heavy chain having the amino acid sequence set forth in SEQ ID NO:33 is set forth in SEQ ID NO:34.

[0376] In certain embodiments, the DNA encoding the heavy chain having the amino acid sequence set forth in SEQ ID NO:35 is set forth in SEQ ID NO:36.

[0377] In certain embodiments, the DNA encoding the heavy chain having the amino acid sequence set forth in SEQ ID NO:52 is set forth in SEQ ID NO:53.

[0378] In certain embodiments, the DNA encoding the heavy chain having the amino acid sequence set forth in SEQ ID NO:56 is set forth in SEQ ID NO:57.

[0379] In still another aspect, the present invention provides a vector comprising the nucleotide composition of the present invention.

[0380] In certain embodiments, the vector is a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.

[0381] In still another aspect, the present invention provides a host cell comprising the vector of the present invention.

[0382] In some embodiments, the host cell containing the vector is obtained by transformation of the vector.

[0383] In some embodiments, the host cell is a bacterial, yeast or mammalian cell.

[0384] In some embodiments, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells.

[0385] In another aspect, the present invention provides a method for preparing the isolated antibody or antigen-binding portion thereof described in the present invention, the method comprising expressing the antibody or antigen-binding fragment thereof in the host cell described in the present invention, and isolating the antibody or antigen-binding fragment thereof.

[0386] In yet another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of the above-described isolated antibody or antigen-binding portion thereof, and a pharmaceutically acceptable excipient.

[0387] In still another aspect, the present invention provides a reagent for measuring LIF in a biological sample, comprising the above-mentioned isolated antibody or antigen-binding portion thereof.

[0388] In certain embodiments, the biological sample is blood, serum, urine, biopsy material, tumor, or any tissue suspected of containing abnormal LIF levels.

[0389] In another aspect, the present invention provides a method for inhibiting LIF expression and / or blocking LIF activity, comprising administering to a patient in need thereof an effective therapeutic dose of the above-mentioned isolated antibody or antigen-binding portion thereof, and / or the above-mentioned pharmaceutical composition.

[0390] In another aspect, the present invention provides use of the above-mentioned isolated antibody or antigen-binding portion thereof, and / or the above-mentioned pharmaceutical composition in a therapeutic drug for inhibiting LIF expression and / or blocking LIF activity.

[0391] In yet another aspect, the present invention provides the above-mentioned isolated antibody or antigen-binding portion thereof, and / or the above-mentioned pharmaceutical composition for inhibiting LIF expression and / or blocking LIF activity.

[0392] In another aspect, the present invention provides a method for treating a disease or condition associated with LIF, the method comprising administering to a patient in need thereof an effective therapeutic dose of the above-described isolated antibody or antigen-binding portion thereof, and / or the above-described pharmaceutical composition. In certain embodiments, the disease associated with LIF is a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the solid tumor comprises glioblastoma, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or prostate cancer.

[0393] In another aspect, the present invention provides the use of the above-mentioned isolated antibody or antigen-binding portion thereof, and / or the above-mentioned pharmaceutical composition in a medicament for treating a disease or condition associated with LIF. In certain embodiments, the disease associated with LIF is a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the solid tumor includes glioblastoma, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer or prostate cancer.

[0394] In yet another aspect, the present invention provides the above-mentioned isolated antibody or antigen-binding portion thereof, and / or the above-mentioned pharmaceutical composition for treating a disease or condition associated with LIF. In certain embodiments, the disease associated with LIF is a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the solid tumor includes glioblastoma, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer or prostate cancer.

[0395] A disease or condition associated with LIF means that blocking LIF and LIRR and / or GP130 can treat, alleviate, ameliorate and / or stabilize the disease or condition.

[0396] In another aspect, the present invention provides a method for detecting LIF in a biological sample, comprising: (i) obtaining a tissue or fluid sample from a subject, (ii) exposing the tissue or fluid sample to the above-mentioned isolated antibody or antigen-binding portion thereof or the above-mentioned reagent; and (iii) detecting LIF bound to the tissue or fluid sample obtained in (ii) and comparing it with LIF bound to a control sample, wherein an increase in the amount of LIF bound compared to the control indicates an abnormal level of LIF production, expression or activation.

[0397] In certain embodiments, the tissue or fluid sample comprises blood, serum, urine, biopsy material, tumor, or any tissue suspected of containing abnormal LIF levels.

[0398] Table 1 Antibody sequence description table of the present invention

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425] Note: All amino acid numbers in CDR and framework regions are annotated according to the EU index of the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH publication No. 91-3242). All sequences do not include signal peptides. aa: amino acid, nt: nucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0426] Figure 1 Demonstrating the ability of the LIF antibody of the present invention to bind to human LIF protein;

[0427] Figure 2 Demonstrating the ability of the LIF antibody of the present invention to bind to mouse LIF protein;

[0428] Figure 3 Demonstrating the ability of the LIF antibody of the present invention to bind to the cynomolgus monkey LIF protein;

[0429] Figure 4 The affinity of the 38E10E1C11 antibody of the present invention for binding to an antigen is shown;

[0430] Figure 5 The ability of the 38E10E1C11 antibody of the present invention to compete with LIFR for binding to human LIF protein is shown;

[0431] Figure 6 The ability of the P36-033 antibody of the present invention to compete with the receptor GP130 for binding to the human LIF protein is shown;

[0432] Figure 7It shows that the 38E10E1C11 antibody and the P36-033 antibody of the present invention cross-react with IL-6 family proteins;

[0433] Figure 8 It was shown that the 38E10E1C11R antibody of the present invention recognized the denatured human LIF protein.

[0434] Fig. 9 Sub-figure A in the figure shows that the 38E10E1C11 antibody of the present invention inhibits the phosphorylation of colon cancer cells (HCT116) induced by human LIF protein; Fig. 9 Sub-figure B in the figure shows that the 38E10E1C11 antibody of the present invention inhibits the phosphorylation of STAT3 of pancreatic cancer cells (KP4) induced by human LIF protein;

[0435] Fig.10 It shows that the 38E10E1C11 antibody of the present invention blocks the activation of STAT3 of pancreatic cancer cell KP4 by human LIF secreted by CT26-hLIF cells;

[0436] Fig.11 The P36-033 antibody of the present invention inhibits the phosphorylation of STAT3 in colon cancer cells (HCT116) induced by human LIF protein;

[0437] Fig.12 It shows that the 38E1E1C11 and P36-033 antibodies of the present invention reverse the proliferation inhibition of M1 cells by LIF;

[0438] Fig.13 It is shown that the 38E10E1C11 antibody of the present invention inhibits the growth of CT26-hLIF cells in BABL / c mice;

[0439] Fig.14 Demonstrating the sensitivity of three human pancreatic cancer cell lines to stimulation with human LIF protein;

[0440] Fig.15 It shows that the 38E10E1C11R antibody of the present invention inhibits the phosphorylation of STAT3 in KP4 cells caused by LIF protein stimulation;

[0441] Fig.16 Sub-figure A in FIG. 1 shows the experimental results of the 38E10E1C11 antibody of the present invention recognizing the full-length human LIF protein and the hybrid LIF protein. Fig.16 Sub-figure B in the figure shows the experimental results that the 38E10E1C11 antibody of the present invention can reverse the inhibition of M1 cell proliferation by full-length human LIF protein and hybrid LIF protein;

[0442] Figures 17A-17D Experimental results showing the antigen binding properties of the humanized anti-LIF antibody of the present invention;

[0443] Fig.18 Experimental results showing the non-specific binding affinity of the humanized anti-LIF antibody of the present invention;

[0444] Fig.19 The experimental results show that the humanized anti-LIF antibody of the present invention competes with LIFR for binding to human LIF protein;

[0445] Fig. 20 The experimental results show that the humanized anti-LIF antibody of the present invention competes with GP130 for binding to human LIF protein;

[0446] Fig.21 Experimental results showing the antigen recognition specificity of the humanized anti-LIF antibody of the present invention;

[0447] Fig. 22 Sub-figure A in the figure shows the experimental results of the present invention showing that the humanized anti-LIF antibody of the present invention recognizes the full-length human LIF protein and the hybrid LIF protein, Fig. 22 Sub-figure B in the figure shows the experimental results of the humanized anti-LIF antibody of the present invention blocking the inhibition of M1 cell proliferation by the full-length LIF protein and the hybrid protein;

[0448] Fig.23 Experimental results showing that the humanized anti-LIF antibody of the present invention inhibits the phosphorylation of STAT3 induced by LIF protein;

[0449] Fig.24 The experimental results show that the humanized anti-LIF antibody of the present invention can reverse the inhibition of M1 cell proliferation by human LIF protein.

[0450] Fig.25 The results showed that humanized anti-LIF antibody detected by HTFR method inhibited the phosphorylation level of STAT3 and the total level of STAT3 induced by LIF protein.

[0451] Fig.26 The ADCC effect of humanized anti-LIF antibody was demonstrated.

[0452] Detailed description

[0453] definition

[0454] To make this specification easier to understand, some terms are first defined. Other definitions are set forth throughout the specification.

[0455] The term "LIF (Leukemia Inhibitory Factor)" as used herein refers to leukemia inhibitory factor. The amino acid sequence of LIF is publicly available (Ref Seq NM_001257135). In some embodiments, LIF can be human, mouse (Ref Seq NM_001039537.2) or cynomolgus monkey LIF (XM_015457518.1). As described elsewhere herein, LIF can be recombinant and / or glycosylated or non-glycosylated.

[0456] The term "antibody" as used herein may include a complete antibody and any antigen-binding fragment thereof (i.e., "antigen-binding portion") or a single chain. In one embodiment, an "antibody" refers to a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions may be further subdivided into hypervariable regions, referred to as complementarity determining regions (CDRs), and more conservative regions, referred to as framework regions (FRs), which are interspersed. Herein, the CDR of the VH region is abbreviated as HCDR, that is, the three CDRs of the VH region can be abbreviated as HCDR1, HCDR2, and HCDR3; the CDR of the VL region is abbreviated as LCDR, that is, the three CDRs of the VL region can be abbreviated as LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0457] The heavy chain of the antibody may or may not contain a terminal lysine (K) or a terminal glycine and lysine (GK). Therefore, any heavy chain sequence and heavy chain constant region sequence provided herein may end with GK or K, or lack K or GK, regardless of the last amino acid of the sequence. This is because the terminal lysine, and sometimes glycine and lysine, are cleaved off during antibody expression.

[0458] Antibodies typically bind specifically to their cognate antigen with high affinity ranging from 10 -7 M to 10 -11 M or less dissociation constant (K D ) is reflected. It is generally considered to be greater than about 10 -6 Any K of M DIndicates non-specific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity and is substantially the same antigen, but does not bind to unrelated antigens with high affinity, high affinity meaning having 10 -7 M or less, preferably 10 -8 M or less, even more preferably 5×10 -9 M or less, and preferably between 10 -8 M and 10 -10 M or smaller K D An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example if it exhibits at least 80%, at least 90%, at least 95%, at least 97% or at least 99% or greater sequence identity to the sequence of the given antigen.

[0459] Immunoglobulins may be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into multiple subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In certain embodiments, the anti-LIF antibodies described herein have a human IgG1 or mouse IgG1 subtype. Immunoglobulins (e.g., human IgG1) exist in several isotypes that differ from each other by at most a few amino acids. For example, "antibodies" may include naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies; and single-chain antibodies.

[0460] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody (e.g., an anti-LIF antibody described herein) include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) a Fd fragment, which consists of the VH and CH1 domains; (iv) a Fv fragment, which consists of the VL and VH domains of one arm of the antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546), which consists of the VH domain; and (vi) isolated complementarity determining regions (CDRs), or (vii) a combination of two or more isolated CDRs, which may be optionally linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be linked to these domains by recombinant methods through synthetic linkers, allowing them to be prepared as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). These single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These and other potential constructs are described in Chan et al.

[0461] Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and screening from these fragments can be used in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0462] The term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region of an antibody of the invention may be replaced by other amino acid residues from the same side chain family, and the retained function of the altered antibody may be tested using the functional assays described herein. Preferably, conservative modifications are no more than 1 or 2 in number.

[0463] Modification of the amino acid sequences described herein is contemplated by the present invention, particularly those of the human heavy chain constant region to reprogram the sequence to a desired allotype, such as that found in Asian populations.

[0464] For example, one or more CDRs or CDR groups of an antibody can be grafted into a framework (such as a human framework) to provide an antibody molecule. The framework region can be a gene sequence of a human reproductive system, or a non-reproductive system. The framework can be reproductive in this way, wherein one or more residues in the framework can be exchanged to match the residues in the most similar human reproductive system framework at a suitable position. Like this, binding members of the present invention can be separated with a VH domain comprising a HCDR group in a human reproductive system framework, for example, a human reproductive system IgG VH framework. Binding members can also have a VL domain comprising a LCDR group, such as in a human reproductive system IgG VL framework.

[0465] The VH and / or VL scaffold residues may be modified as discussed and exemplified herein, such as using site-directed mutagenesis.The VH or VL domains, or binding members of the invention comprise such VL domains.

[0466] Can change in one or more framework regions and / or one or more CDR, described change usually does not cause loss of function, so the binding member that comprises this change aminoacid sequence should keep the ability of binding and / or neutralizing LIF.It can keep and have the same amount of binding and / or neutralizing ability as the binding member that does not carry out change, as measured by analytical method as described herein.The binding member that comprises the aminoacid sequence of such change can have improved ability of binding and / or neutralizing LIF.

[0467] The changes may include replacement of one or more amino acid residues with non-natural or non-standard amino acids, modification of one or more amino acid residues to non-natural or non-standard forms, or insertion of one or more non-natural or non-standard amino acids into the sequence. Examples of the position and number of changes in the sequences of the present invention are described elsewhere herein. Naturally occurring amino acids include 20 "standard" L-amino acids identified by their standard single-letter codes as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E. Non-standard amino acids include any other residues that can be incorporated into the polypeptide backbone or modified from existing amino acid residues. Non-standard amino acids may be naturally occurring or non-naturally occurring. Several naturally occurring non-standard amino acids are known in the art, 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, N-ethylserine, etc. (Voet & Voet, 1995, Biochemistry, 2nd Edition, (Wiley)). Those amino acid residues that are derivatized at their N-α position will only be located at the N-terminus of the amino acid sequence. Typically the amino acids in the present invention are L-amino acids, but may be D-amino acids. Thus variations may include modification with L-amino acids or substitution of D-amino acids for L-amino acids. Formylated, acetylated and / or phosphorylated forms of amino acids are known, and the amino acids of the present invention may be so modified.

[0468] The amino acid sequence in the binding members and antibody domains of the present invention may include the non-natural or non-standard amino acids described above. Non-standard amino acids (such as D-amino acids) may be incorporated into the amino acid sequence during the synthesis process, or by modification or replacement of "original" standard amino acids after amino acid synthesis.

[0469] The use of non-standard and / or non-naturally occurring amino acids increases the diversity of structure and function and can increase the potential to achieve the desired LIF binding and neutralization properties in the binding members of the present invention. In addition, D-amino acids and analogs have been shown to have better pharmacokinetic properties than standard L-amino acids due to the in vivo degradation of polypeptides with L-amino acids after administration to animals such as humans.

[0470] The generation of new VH or VL regions of the invention with CDR-derived sequences can employ random mutagenesis of one or more selected VH and / or VL genes to generate mutants within all variable regions. Such techniques are described in Gram et al. (Gram et al., 1992, Proc. Natl. Acad. Sci., USA, 89: 3576-3580), which uses error-prone PCR. In some embodiments, one or more amino acid substitutions are made within all variable regions or CDR groups.

[0471] Another method that can be used is targeted mutagenesis of the CDR regions of the VH or VL genes. Such methods are disclosed by Barbas et al. (Barbas et al., 1994, Proc. Natl Acad. Sci., USA, 91: 3809-3813) and Schier et al. (Schier et al., 1996, J. Mol. Biol. 263: 551-567).

[0472] All methods described above are known in the art, and those skilled in the art will be able to use such methods and adopt routine methods in the art to provide binding members of the present invention.

[0473] Variant domain amino acid sequence variants of any VH and VL domains of the specific sequences disclosed herein can be used according to the present invention, as discussed. Specific variants may include one or more amino acid sequence changes (additions, deletions, substitutions and / or insertions of amino acid residues). In certain embodiments, the variant has less than about 17, less than 9, or less than 5 such changes.

[0474] As indicated above, CDR amino acid sequences substantially as described herein may be carried as CDRs in a human antibody variable region or a substantial portion thereof. HCDR3 sequences substantially as described herein represent embodiments of the invention, each of which may be carried as CDRs in a human antibody variable region or a substantial portion thereof, optionally in combination with HCDR1, HCDR2, LCDR1, LCDR2 and LCDR3 of the invention.

[0475] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a specific epitope, or an antibody composition, wherein all antibodies in the antibody composition display a single binding specificity and affinity for a specific epitope. Typically, these monoclonal antibodies will be derived from a single antibody encoding cell or nucleic acid, and will proliferate without intentionally introducing any sequence changes. Therefore, the term "human monoclonal antibody" refers to a monoclonal antibody with a variable region and an optional constant region derived from a human germline immunoglobulin sequence. In one embodiment, a human monoclonal antibody is produced by, for example, a hybridoma obtained by fusing a B cell obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome comprising a human heavy chain transgene and a light chain transgene) and an immortal cell. The term "mAb" refers to a monoclonal antibody.

[0476] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal to human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, such as transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies include variable and constant regions that utilize specific human germline immunoglobulin sequences and are encoded by germline genes, but include subsequent rearrangements and mutations that occur during, for example, antibody maturation. As known in the art (e.g., see Lonberg (2005) Nature Biotech. 23 (9): 1117-1125), the variable region contains antigen binding domains encoded by different genes that are rearranged to form antibodies specific for foreign antigens. In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (called somatic mutations or hypermutations) to increase the affinity of the antibody for foreign antigens. The constant region will further change in response to antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid sequences encoding light and heavy chain immunoglobulin polypeptides in response to antigen may not be identical to the original germline sequences, but will be substantially identical or similar (i.e., have at least 80% identity).

[0477] "HuMAb" refers to an antibody having a variable region in which the framework region and the CDR region in the variable region are all derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The antibodies described herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0478] A "humanized" antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced by corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced by amino acids from human immunoglobulins, while some, most or all of the amino acids in one or more CDR regions have not changed. Small additions, deletions, insertions, substitutions or modifications of amino acids may be tolerated as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

[0479] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0480] The functional antibody fragments of the present invention include any functional fragments whose half-life is increased by chemical modification such as PEGylation or incorporation into liposomes.

[0481] The antibodies of the present invention include bispecific antibodies. Bispecific or bifunctional antibodies form the second generation of monoclonal antibodies in which two different variable regions are combined into the same molecule (Holliger and Bohlen, 1999 Cancer and metastasis rev. 18: 411-419). Their applications in the diagnosis and treatment fields have been described from their ability to recruit new effector functions or target some molecules on the surface of tumor cells. Where bispecific antibodies are used, these can be conventional bispecific antibodies, which can be manufactured in various ways (Holliger P. & Winter G. Current Opinion Biotechnol. 4, 446-449: 1993), such as chemical preparation, or hybridomas from hybridization, or can be any of the bispecific antibody fragments mentioned above. These antibodies can be obtained by chemical methods or somatic cell methods, but similarly and preferably genetic engineering methods, which allow heterodimerization to be implemented, which is conducive to the purification process of the obtained antibodies. Examples of bispecific antibodies include those of the BiTE™ approach, in which the binding domains of two antibodies with different specificities can be used and directly linked by a short flexible peptide. This combines two antibodies on a short single polypeptide chain. Diabodies and scFcs are constructed without the Fc region, using only the variable region, potentially reducing the effect of anti-idiotypic responses.

[0482] The bispecific antibody can be constructed as whole IgG, bispecific (Fab')2, (Fab')PEG, diabody or other bispecific scFv. Further, two bispecific antibodies can be linked to form a tetravalent antibody using conventional methods known in the art.

[0483] Relative to bispecific full antibodies, bispecific diabodies are also particularly useful because they can be easily constructed and expressed in E. coli. Using phage display libraries (WO1994 / 13804, diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificity can be easily selected. If one arm of the diabody is kept constant, a library is then prepared in which the other arms are mutated to select antibodies of appropriate specificity. Bispecific full antibodies can be prepared by different engineering methods, which are described in Ridgeway et al. (Ridgeway, JBB et al., Protein Eng.9, 616-621, 1996) or WO1996 / 27011, WO1998 / 50431 and WO2006 / 028936.

[0484] A "modified heavy chain constant region" refers to a heavy chain constant region comprising constant domains CH1, hinge, CH2, and CH3, wherein one or more constant domains are from different isotypes (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the modified constant region comprises a human IgG2 CH1 domain and a human IgG2 hinge and a human IgG1 CH3 domain fused to a human IgG1 CH2 domain. In certain embodiments, these modified constant regions also include amino acid modifications within one or more domains relative to the wild-type amino acid sequence.

[0485] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region genes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibodies).

[0486] "Isotype" refers to naturally occurring variants within a particular isotype group that differ by a few amino acids (eg, see Jefferis et al. (2009) mAbs 1:1). The antibodies described herein may be of any isotype.

[0487] Unless otherwise indicated elsewhere herein, all amino acid numbering is according to the EU index of the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed., US Department of Health and Human Services, NIH Publication No. 91-3242).

[0488] "An antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0489] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free from other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to LIF is substantially free from antibodies that specifically bind to antigens other than LIF). However, an isolated antibody that specifically binds to an epitope of LIF may have cross-reactivity to other LIF proteins from different species.

[0490] "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event derived therefrom. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (e.g., ADCC and antibody-dependent cell-mediated phagocytosis (ADCP)), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). These effector functions generally require the combination of an Fc region with a binding domain (e.g., an antibody variable domain).

[0491] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). The various properties of human FcγRs are summarized in Table A. Most innate effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans), but do not express inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered to bind to an activating Fc receptor type equivalent to murine IgG2a.

[0492] Table A. Properties of human FcγRs

[0493]

[0494] "Hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region that connects the CH1 domain and the CH2 domain and includes the upper, middle and lower parts of the hinge (Roux et al., J. Immunol. 1998

[0495] 161:4083). The hinge provides a variable flexibility between the binding and effector regions of the antibody and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions.

[0496] The term "hinge" includes wild-type hinges and variants thereof (e.g., non-naturally occurring hinges or modified hinges). For example, the term "IgG2 hinge" includes wild-type IgG2 hinges and variants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or at most 5, 4, 3, 2 or 1 mutations (e.g., substitutions, deletions or additions).

[0497] The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge in the heavy chain constant domain. The term "CH1 domain" includes wild-type CH1 domains and variants thereof (e.g., non-naturally occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes wild-type CH1 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or up to 5, 4, 3, 2 or 1 mutation (e.g., substitution, deletion or addition). Exemplary CH1 domains include CH1 domains with mutations that change the biological activity (e.g., ADCC, CDC or half-life) of the antibody.

[0498] The term "CH2 domain" refers to the heavy chain constant region connecting the hinge in the heavy chain constant domain to the CH3 domain. The term "CH2 domain" includes wild-type CH2 domains and variants thereof (e.g., non-naturally occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wild-type CH2 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or up to 5, 4, 3, 2 or 1 mutations (e.g., substitutions, deletions or additions). Exemplary CH2 domains include CH2 domains with mutations that alter the biological activity of the antibody (e.g., ADCC, CDC or half-life).

[0499] The term "CH3 domain" refers to the heavy chain constant region that is the C-terminal end of the CH2 domain in the heavy chain constant domain. The term "CH3 domain" includes wild-type CH3 domains and variants thereof (e.g., non-naturally occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wild-type CH3 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or at most 5, 4, 3, 2 or 1 mutations (e.g., substitutions, deletions or additions). Exemplary CH3 domains include CH3 domains with mutations that change the biological activity (e.g., ADCC, CDC or half-life) of the antibody.

[0500] "CL domain" refers to the constant domain of the light chain. The term "CL domain" includes wild-type CL domain and variants thereof.

[0501] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions; native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions and natural variants thereof. Native sequence Fc includes multiple isotypes of Fc (e.g., see Jefferis et al. (2009) mAbs 1:1).

[0502] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., LIF) to which an immunoglobulin or antibody specifically binds. Epitopes within a protein antigen can be formed from both continuous amino acids (usually linear epitopes) or non-continuous amino acids juxtaposed by the tertiary folding of the protein (usually conformational epitopes). When exposed to a denaturing solvent, epitopes formed from continuous amino acids are usually, but not always, retained, while epitopes formed by tertiary folding are usually lost when treated with a denaturing solvent. In a unique spatial conformation, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. Methods for determining epitope binding by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation analysis, in which overlapping or continuous peptides (e.g., from LIF) are tested for reactivity with a given antibody (e.g., anti-LIF antibody). Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as x-ray crystallography, 2-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, ed. (1996)).

[0503] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of another antibody to a target. Known competition experiments (such as those described in the Examples) can be used to determine whether two antibodies compete with each other for binding to a target, i.e., whether one antibody inhibits the binding of another antibody to a target and the degree of inhibition. In certain embodiments, an antibody competes with another antibody for binding to a target and inhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the binding. The degree of inhibition or competition may vary depending on which antibody is a "blocking antibody" (i.e., a cold antibody that is first incubated with the target). Competition assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101 / pdb.prot4277 or Chapter 11 of "Using Antibodies", Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance).

[0504] Other competitive binding assays include: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeling assay, solid phase direct labeling sandwich analysis (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeling RIA using 1-125 labeling (see Morel et al.,

[0505] Mol. Immunol. 25(1):7 (1988); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0506] As used herein, the term "Kassoc" or "Ka" is intended to refer to the association rate constant of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" is intended to refer to the dissociation rate constant of a particular antibody-antigen interaction. D " refers to the equilibrium dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed in molar concentration (M). The K of an antibody can be determined using methods well established in the art. D Determine the K value of the antibody D A preferred method is by using surface plasmon resonance, preferably using a biosensor system, such as Surface plasmon resonance system or flow cytometry or Scatchard analysis.

[0507] The term "EC50" refers to the concentration of an antibody or antigen-binding portion thereof that induces a 50% maximal response, ie, a response halfway between the maximal response and baseline, in the context of an in vitro or in vivo assay using the antibody or antigen-binding fragment thereof.

[0508] The term "IC50" refers to the concentration of a binding member that reduces a biological response to 50% of its maximum value in functional assays, in nM. In ligand-binding studies, IC50 is the concentration that reduces receptor binding to 50% of the maximum specific binding level. IC50 can be obtained by plotting the percentage of the maximum biological activity response as a function of the log of the binding member concentration, and using a software program such as Origin (OriginLab Software, Northampton, Massachusetts, USA) to fit an S function to the data to generate an IC50 value. The titer can be determined or measured using one or more analytical methods known to those skilled in the art and / or described or referenced herein. The neutralization titer of a binding member can be expressed as a geometric mean.

[0509] As used herein, the term "naturally occurring" when applied to a target refers to the fact that the target can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring.

[0510] "Polypeptide" refers to a chain of at least two consecutively linked amino acid residues, and the length of the chain is unlimited. One or more amino acid residues in a protein may contain modifications, such as (but not limited to) glycosylation, phosphorylation or disulfide bonds. A "protein" may contain one or more polypeptides.

[0511] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA. "Conservative sequence modifications" of the sequences described in the SEQ ID NOs described herein are also provided, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of antibodies encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the SEQ ID NOs described herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which amino acid residues are replaced with amino acid residues with similar side chains. Families of amino acid residues with similar side chains are already defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0512] For nucleic acids, the term "substantial homology" means that two nucleic acids, or designated sequences thereof, are identical in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). Alternatively, substantial homology exists when segments hybridize under selective hybridization conditions to the complement of a strand.

[0513] With respect to polypeptides, the term "substantial homology" means that two polypeptides or designated sequences thereof are identical in at least about 80% of their amino acids, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% when optimally aligned and compared (with appropriate insertions or deletions of nucleotides).

[0514] The % identity between two sequences varies with the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions x 100), wherein optimal alignment is determined by taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0515] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) algorithm incorporated into the ALIGN program, version 2.0, using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0516] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to perform searches against public databases, e.g., to identify related sequences. These searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0517] These nucleic acids may be present in intact cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified by standard techniques to remove other cellular components or other contaminants such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art. See F. Ausubel et al., eds. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0518] Nucleic acids (e.g., cDNA) can be mutated according to standard techniques to provide gene sequences. For coding sequences, these mutations may affect the amino acid sequence as desired. Specifically, DNA sequences substantially homologous to or derived from natural V sequences, D sequences, J sequences, constant sequences, switch sequences, and other such sequences described herein are contemplated.

[0519] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes operably connected thereto. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably, because plasmids are the most commonly used forms of vectors. However, the present invention also includes other forms of expression vectors that provide equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0520] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that these terms are intended to refer not only to a specific individual cell, but also to the progeny of such a cell. Because mutations or environmental influences may cause certain changes in subsequent generations, the progeny may not actually be the same as the parent cell, but are still included in the scope of the term "host cell" as used herein.

[0521] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide or hapten. The antigen may be LIF or a fragment thereof.

[0522] As used herein, the terms "inhibit" or "block" (eg, in reference to inhibition / blocking of LIF binding or activity) are used interchangeably and encompass both partial and complete inhibition / blocking.

[0523] As used herein, "cancer" refers to a broad class of diseases characterized by uncontrolled abnormal cell growth in the body. Malignant tumors or cells may form as a result of unregulated cell division, which invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0524] As used herein, the term "treatment" or "treating" refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, to reverse, alleviate, ameliorate, inhibit or slow or prevent the progression, development, severity or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease. Prevention refers to the administration to a subject who does not have the disease to prevent the disease from occurring, or if the disease occurs, to minimize its effects.

[0525] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, can promote disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability caused by the disease. A "prophylactically effective amount" or "prophylactically effective dose" of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of contracting a disease or suffering a recurrence of the disease, inhibits the development or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the development or recurrence of a disease can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that are predictive of efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

[0526] The terms "patient" and "subject" refer to any human or non-human animal receiving a prophylactic or therapeutic treatment. For example, a subject with cancer can be treated using the methods and compositions described herein. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. Example

[0527] Example 1 Screening and identification of anti-human LIF monoclonal antibodies

[0528] 1.1 Preparation of anti-human LIF monoclonal antibody 38E10E1C11 by hybridoma method

[0529] According to the monoclonal antibody preparation method (Kohler and Milstein (1975) Nature 256: 495), BABL / c mice were immunized with recombinant human LIF protein (purchased from Sino Biological). The first immunization used 25 μg of recombinant human LIF protein and an equal volume of Freund's complete adjuvant, which was injected subcutaneously at multiple points on the back. Four weeks later, the second immunization used Freund's incomplete adjuvant plus 25 μg of recombinant human LIF protein. After 20 days, the mouse serum antibody titer was detected by indirect ELISA. After an interval of 2-3 weeks, 50 μg of recombinant human LIF protein was injected intraperitoneally to boost immunization. After 3 days, the animals were killed and spleen cells were taken for fusion.

[0530] Mouse myeloma cells SP2 / 0 in the logarithmic growth phase were counted, and spleen cell suspension of immunized mice was prepared at the same time. The spleen cells and SP2 / 0 cells were fused with 50% PEG according to the conventional method. The fused cells were added to a 96-well plate of trophoblast cells (peritoneal macrophages of BABL / c mice aged six weeks) and screened and cultured in DMEM containing 1% HAT and 20% fetal bovine serum. When the clones grew to 1 / 3 of the bottom of the plate, the culture supernatant was collected. ELISA plates were coated with recombinant human LIF protein, and anti-LIF antibodies in the culture supernatant were detected by indirect ELISA method, clones secreting anti-human LIF antibodies were screened, and further monoclonal production was performed by limiting dilution method to obtain a cell line that can stably secrete high-affinity anti-human LIF monoclonal antibodies. The secreted antibodies were labeled as 38E10E1C11. After determination, the full-length gene sequences encoding the light chain and heavy chain of the 38E10E1C11 antibody were shown in SEQ ID NO:42 and SEQ ID NO:44, respectively, and the corresponding full-length amino acid sequences of the light chain and heavy chain of the 38E10E1C11 antibody were shown in SEQ ID NO:41 and SEQ ID NO:43, respectively; the variable region gene sequences encoding the light chain and heavy chain of the 38E10E1C11 antibody were shown in SEQ ID NO:76 and SEQ ID NO:77, respectively, and the corresponding amino acid sequences of the variable regions of the light chain and heavy chain of the 38E10E1C11 antibody were shown in SEQ ID NO:74 and SEQ ID NO:75, respectively. NO:75; according to the Kabat coding rules, the amino acid sequence of LCDR1 of the 38E10E1C11 antibody is shown in SEQ ID NO:1, the amino acid sequence of LCDR2 is shown in SEQ ID NO:2, the amino acid sequence of LCDR3 is shown in SEQ ID NO:3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is shown in SEQ ID NO:45, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:6. The immunoglobulin type and subtype of the antibody were identified (the result was IgG1 subclass, k-type light chain).

[0531] After obtaining a hybridoma cell line that can stably secrete antibodies, the cells are acclimated using Thermo Fisher's CDhybridoma serum-free medium to adapt them to serum-free suspension shaking culture, and then the serum-free medium is used to express and purify the antibodies.

[0532] 1.2 Preparation of anti-human LIF monoclonal antibody P36-033 using phage display technology

[0533] BABL / c mice were immunized with recombinant human LIF protein. For the first immunization, 25 μg pure recombinant human LIF protein and an equal volume of Freund's complete adjuvant were used for subcutaneous injection at multiple points on the back. Four weeks later, the second immunization was performed with Freund's incomplete adjuvant plus 25 μg recombinant human LIF protein. The mouse serum antibody titer was detected by indirect ELISA 20 days later. After an interval of 2-3 weeks, 50 μg recombinant human LIF protein was injected intraperitoneally for booster immunization. The animals were killed 3 days later to obtain spleen cells. Total RNA of spleen cells was extracted using Invitrogen's TRIZOL Reagent, reverse transcribed using Invitrogen cDNA reverse transcription kit, and cDNA was synthesized. Antibody genes were amplified using mouse light and heavy chain variable region degenerate primers, constructed into phage display vectors, and phage antibody libraries were constructed. The thermo's fully automatic magnetic bead sorting system was used to screen the phage antibody library. Phage ELISA was used to select E. coli clones that could bind to recombinant human LIF protein, and the antibody gene sequence was determined. Further, an antibody labeled as P36-033 was obtained through ELISA and cell activity identification. The full-length gene sequences of the light chain and heavy chain of P36-033 are shown in SEQ ID NO:55 and SEQ ID NO:57, respectively, and the corresponding full-length amino acid sequences of the light chain and heavy chain of the P36-033 antibody are shown in SEQ ID NO:54 and SEQ ID NO:56, respectively; the variable region gene sequences encoding the light chain and heavy chain of the P36-033 antibody are shown in SEQ ID NO:72 and SEQ ID NO:73, respectively, and the corresponding amino acid sequences of the variable regions of the light chain and heavy chain of the P36-033 antibody are shown in SEQ ID NO:82 and SEQ ID NO:83, respectively; according to the Kabat encoding rule, the amino acid sequence of LCDR1 of the P36-033 antibody is shown in SEQ ID NO:66, the amino acid sequence of LCDR2 is shown in SEQ ID NO:67, the amino acid sequence of LCDR3 is shown in SEQ ID NO:68, and the amino acid sequence of HCDR1 is shown in SEQ ID NO: The amino acid sequence of HCDR2 is shown in SEQ ID NO:69, the amino acid sequence of HCDR3 is shown in SEQ ID NO:71.

[0534] 1.3 Expression and purification of positive control antibody 5D8

[0535] According to the report of patent document WO 2018 / 115960 A1, 5D8 antibody is an antibody that blocks the binding of LIF protein to its receptor GP130. With reference to the sequences in the patent document, the present invention synthesized multiple gene sequences, performed different light and heavy chain combinations and pairings, and constructed a full-length antibody in the form of human IgG1. It was finally found that one of the pairings had the best LIF protein binding activity and could block the binding of recombinant human LIF protein to human GP130 protein. After cell activity verification, it could block the phosphorylation of STAT3 of HCT116 cells by recombinant human LIF protein. Therefore, the present invention named it 5D8 and used it as a positive control antibody in subsequent experiments. After measurement, the full-length gene sequences encoding the heavy chain and light chain of 5D8 antibody are shown as SEQ ID NO:63 and SEQ ID NO:65, respectively, and the corresponding full-length amino acid sequences of the heavy chain and light chain of 5D8 antibody are shown as SEQ ID NO:62 and SEQ ID NO:64, respectively; the variable region gene sequences encoding the heavy chain and light chain of 5D8 antibody are shown as SEQ ID NO:80 and SEQ ID NO:81, respectively. NO:81, and the amino acid sequences of the variable regions of the heavy chain and light chain of the corresponding 5D8 antibody are shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.

[0536] Example 2 Binding test of anti-human LIF antibody and human LIF protein

[0537] Recombinant human LIF protein was diluted to 1 μg / ml, coated on the ELISA plate, 100 μL protein was added to each well, and the plate was incubated overnight at 4°C. The next day, the plate was removed, the liquid was discarded, washed three times with PBST, blocked at room temperature for one hour with PBS containing 2% BSA, washed three times with PBST, anti-human LIF antibodies 38E10E1C11, 5D8, and P36-033 of different concentrations were added, incubated at room temperature for one hour, the liquid was discarded, washed four times with PBST, HRP-labeled goat anti-mouse Fab antibody or goat anti-human FC antibody was added, incubated at room temperature for one hour, the liquid was discarded, washed four times with PBST, TMB colorimetric solution was added, incubated at room temperature for 10 minutes, 2 mol / L concentrated sulfuric acid was added to terminate the color development, and the absorbance at 450 nm was read by an ELISA reader. For details, see Figure 1 The results showed that the binding activity of 38E10E1C11 and P36-033 to human LIF protein was stronger than that of 5D8.

[0538] Example 3 Binding test of anti-human LIF antibody and mouse LIF protein

[0539] Recombinant mouse LIF (purchased from ACRO Biosystems) protein was diluted to 1 μg / mL and coated on an ELISA plate. 100 μL of protein was added to each well and incubated in a refrigerator at 4°C overnight. The ELISA plate was taken out on the next day, the liquid was discarded, the plate was washed three times with PBST, and PBS containing 2% BSA was added to block at room temperature for one hour. The plate was washed three times with PBST, and different concentrations of anti-human LIF antibodies 38E10E1C11, 5D8, and P36-033 were added. The plate was incubated at room temperature for one hour. The liquid was discarded, the plate was washed four times with PBST, and HRP-labeled goat anti-mouse Fab antibody or goat anti-human FC antibody was added. The plate was incubated at room temperature for one hour. The liquid was discarded, the plate was washed four times with PBST, and TMB colorimetric solution was added. The plate was incubated at room temperature for 10 minutes. 2 mol / L concentrated sulfuric acid was added to terminate the color development. The absorbance at 450 nm was read by an ELISA reader. The results are shown in Figure 2 The results showed that the 38E10E1C11 antibody had no binding activity to the mouse LIF protein. P36-033 had binding activity to the mouse LIF protein, but the binding activity was weaker than that of the 5D8 antibody.

[0540] Example 4 Binding test of anti-human LIF antibody and cynomolgus monkey LIF protein

[0541] Recombinant monkey LIF protein (purchased from Sino Biological) was diluted to 0.5 μg / mL and coated on an ELISA plate. 100 μL of protein was added to each well and incubated overnight in a 4°C refrigerator. The next day, the ELISA plate was removed, the liquid was discarded, the plate was washed three times with PBST, and PBS containing 2% BSA was added to block at room temperature for one hour. The plate was washed three times with PBST, and different concentrations of anti-human LIF antibodies 38E10E1C11, 5D8, and P36-033 were added. The plate was incubated at room temperature for one hour. The liquid was discarded, the plate was washed four times with PBST, and HRP-labeled goat anti-mouse Fab antibody or goat anti-human Fc antibody was added. The plate was incubated at room temperature for one hour. The liquid was discarded, the plate was washed four times with PBST, and TMB colorimetric solution was added. The plate was incubated at room temperature for 10 minutes. 2 mol / L concentrated sulfuric acid was added to terminate the color development. The absorbance at 450 nm was read by an ELISA reader. The results are shown in Figure 3 The results showed that the binding activity of 38E10E1C11 and P36-033 was better than that of 5D8.

[0542] Example 5 Anti-human LIF antibody affinity determination

[0543] KinExA 4000 was used to measure the affinity of the purified monoclonal antibody to the recombinant human LIF protein. 30ug of 38E10E1C11 antibody was added to 200mg PMMAhard beads, and the coating solution was added to 1.0mL. The buffer composition was 1×PBS, pH7.4, 0.02% NaN3. Ensure that the beads are completely suspended in the solution. Rotate the beads at room temperature for 2 hours. The beads were naturally precipitated or centrifuged briefly at low speed, the supernatant was removed, and blocked with PBS containing 1% BSA for 1 hour. Prepare 15mL of 300pM antigen solution and 15mL of 240pM Ab2 (38E10E1C11) solution. Take 0.6mL of 300pM antigen and 0.6mL of 240pM antibody Ab2 (38E10E1C11) and put them into different sample tubes. Mix the samples in the two tubes thoroughly and combine them into one tube. At this time, the concentration of the antigen is 150pM, and the concentration of the antibody Ab2 (38E10E1C11) is 120pM. Place them in the corresponding position in the tube rack. Prepare 16 groups, and the incubation time of each group of samples is different. Add 1μg / ml Streptavidin Protein, DyLight 650 solution, test on the machine, and set the incubation time to 24 hours. In KinExatm Pro software, use the unknown ligand model to calculate the equilibrium dissociation constant (Kd) for n-curve analysis. The results are detailed in Figure 4 The results showed that the affinity of 38E10E1C11 monoclonal antibody for human LIF protein was 4.52x10 -12 M.

[0544] Example 6P36-033, 38E10E1C11 mAb competes with LIFR for binding to human LIF protein

[0545] The recombinant human LIF protein was coated on the ELISA plate at a concentration of 1 μg / mL, and 0.6125 μg / mL of LIFR (with human F C Different concentrations of LIF antibodies 38E10E1C11, P36-033, and 38E10E1C11R (SEQ ID NOs: 41 and 43) recombinantly expressed by CHO cells were added at 50 μL / well and incubated at room temperature for 2 hours. After washing four times with PBST, HRP-labeled goat anti-human F C The secondary antibody was incubated at room temperature for 1 hour, and after washing four times with PBST, TMB colorimetric solution was added for color development at room temperature for 10 minutes. The absorbance at 450 nm was read by an enzyme reader, and the data were analyzed and plotted using Origin pro 9 software. Figure 5The results showed that 38E10E1C11 mAb and 38E10E1C11R could inhibit the binding of human LIF to human LIFR, while P36-033 could not inhibit the binding of human LIF to human LIFR.

[0546] Example 7P36-033, 38E10E1C11 mAb competes with GP130 for binding to human LIF protein

[0547] The recombinant human LIF protein was coated on the ELISA plate at a concentration of 1 μg / mL, and 20 μg / mL GP130 (fused with human Fc) protein was added at 50 μL / well. At the same time, different concentrations of P36-033 antibody or different concentrations of 38E10E1C11 antibody were added at 50 μL / well.

[0548] The wells were set up, and the control wells with only antibody but no GP130 were set up. They were incubated at room temperature for 2 hours. After washing with PBST four times, HRP-labeled goat anti-human Fc secondary antibody was added and incubated at room temperature for 1 hour. After washing with PBST four times, TMB color development solution was added and color was developed at room temperature for 10 minutes. The absorbance value at 450nm was read by an enzyme reader, and the data were analyzed and plotted using Origin pro 9 software. Figure 6 The results showed that P36-033 and 38E10E1C11 could inhibit the binding of human LIF to human GP130.

[0549] Example 8 Specific Detection of 38E10E1C11 mAb

[0550] Human LIF, human IL-6, human OSM, and human CNTF (all purchased from Sino Biological) were coated on the ELISA plate at a concentration of 1 μg / mL, and different concentrations of LIF antibodies 38E10E1C11, P36-033, and 5D8 were added and incubated at room temperature for 1 hour. After washing four times with PBST, HRP-labeled goat anti-mouse Fab secondary antibody was added and incubated at room temperature for 1 hour. After washing four times with PBST, TMB colorimetric solution was added for color development at room temperature for 10 minutes. The absorbance value at 450 nm was read by an ELISA reader, and the data were analyzed and plotted using Origin pro 9 software. Figure 7 The results showed that 38E10E1C11 monoclonal antibody and P36-033 monoclonal antibody only bound to human LIF protein but not to human IL-6, OSM, and CNTF. In addition to binding to human LIF protein, 5D8 antibody also bound to human OSM and human CNTF proteins.

[0551] Example 9 38E10E1C11 mAb can be used for western blot detection of human LIF protein

[0552] Recombinant human LIF protein was diluted to Figure 8The concentrations shown in the table above were used, or the supernatant of CT26 and CT26-hLIF cells cultured for three days was added with 5XSDS-PAGE loading buffer, boiled for 10 minutes, and then 10 μL of the sample was taken for SDS-PAGE electrophoresis, and then the electrophoresis bands were transferred to a PVDF membrane for western blot detection. The primary antibody used for detection was 38E10E1C11 antibody, and the antibody concentration was 1 μg / mL. After incubation at room temperature for 2 hours, TBST buffer was washed three times, and a 1:3000 diluted HRP-labeled goat anti-mouse secondary antibody was added, incubated at room temperature for 2 hours, incubated at room temperature for 1 hour, TBST buffer was washed three times, and enhanced chemiluminescence solution (Perice) was added for incubation. The cells were detected and photographed using an Amersham Imager 600 ultra-sensitive multifunctional imager. For details, see the results. Figure 8 The results showed that 38E10E1C11 mAb can be used for western blot detection of human LIF protein.

[0553] Example 10 Detection of cell activity of anti-human LIF antibody

[0554] 10.1 STAT3 activation inhibition assay in HCT116 cells

[0555] After HCT116 cells were digested and centrifuged, the cells were resuspended in 1 mL, 5x10 5 cells / well, spread on a 12-well plate. Culture overnight at 37°C, 5% CO2. The next day, discard the original culture medium, add cell culture medium containing 100 ng / mL recombinant human LIF protein and different concentrations of anti-LIF antibodies, set up control wells without recombinant human LIF protein and only with recombinant human LIF protein without antibodies, and incubate in a 37°C incubator for 30 minutes. Then remove the culture medium, add 100uL 1x lysis buffer to each well of the 12-well plate, and lyse on ice for 30 minutes. Transfer the lysate to a 1.5mL centrifuge tube, centrifuge at 13,000rpm for 10 minutes, and collect the supernatant. Take the supernatant for western blot detection of STAT3 phosphorylation. For details of the results, see Fig. 9 The subgraph A and Fig.11 The results show that 38E10E1C11 antibody and P36-033 antibody can inhibit the phosphorylation of STAT3 in HCT116 cells induced by human LIF protein.

[0556] 10.2 Detection of LIF antibody activity by KP4 cell STAT3 activation inhibition assay

[0557] After KP4 cells were digested and centrifuged, the cells were resuspended in 1 mL, 5x10 5Cells / well were plated in 12-well plates. Incubate overnight at 37°C and 5% CO2. The next day, the original culture medium was discarded, and cell culture medium containing 50ng / mL recombinant human LIF protein and different concentrations of anti-LIF antibodies was added. Control wells without recombinant human LIF protein and only with recombinant human LIF protein without antibodies were set up, and incubated in a 37°C incubator for 30 minutes. Then the culture medium was removed, and 1x cell lysis buffer was added to the cells, 100uL per well, and lysed on ice for 30 minutes. The lysate was transferred to a 1.5mL centrifuge tube, centrifuged at 13,000rpm for 10 minutes, and the supernatant was collected. The supernatant was taken for western blot to detect the phosphorylation of STAT3. Fig. 9 The results in subfigure B show that the 38E10E1C11 antibody can inhibit the phosphorylation of STAT3 in KP4 cells induced by human LIF protein.

[0558] 10.3KP4 cell STAT3 activation inhibition assay to detect LIF antibody activity

[0559] After KP4 cells were digested and centrifuged, the cells were resuspended in 1 mL, 5x10 5 Cells / well were plated in 12-well plates. Incubate overnight at 37°C and 5% CO2. On the second day, discard the original culture medium, prepare different concentrations of anti-LIF antibody and CT26-hLIF cell culture medium in a volume ratio of 1:1 and add them to the cells. Set up a control well for CT26 culture supernatant and incubate in a 37°C incubator for 30 minutes. Then remove the culture medium, add 1x cell lysis buffer to the cells, 100uL per well, and lyse on ice for 30 minutes. Transfer the lysate to a 1.5mL centrifuge tube, centrifuge at 13,000rpm for 10 minutes, and collect the supernatant. Take the supernatant for western blot to detect the phosphorylation of STAT3. Fig.10 It was shown that the 38E10E1C11 antibody of the present invention blocks the activation of STAT3 of pancreatic cancer cell KP4 by human LIF secreted by CT26-hLIF cells.

[0560] 10.4M1 cell proliferation assay to detect LIF antibody activity

[0561] After M1 centrifugation, the cells were washed twice with RPMI1640 medium at 2x10 5 / mL density inoculated 96-well plates, 100ul cells were inoculated in each well, and culture medium containing 10ng / mL recombinant human LIF protein and different concentrations of anti-LIF antibodies was added to make the final volume of each well 200uL. At the same time, control wells without LIF were set up, and cultured in a 37℃ incubator for 72 hours. CCK-8 was added to detect cell proliferation. The results are detailed in Fig.12The results showed that both monoclonal antibody 38E10E1C11 and monoclonal antibody P36-033 could reverse the proliferation inhibition of M1 cells by human LIF protein.

[0562] Example 11 Detection of anti-tumor activity of anti-human LIF antibody in vivo

[0563] ELISA detection showed that the 38E10E1C11 antibody had no cross-reaction with mouse LIF protein. In order to evaluate the in vivo activity, it was necessary to construct a CT26 cell line that overexpressed human LIF protein. According to literature reports, human LIF protein can bind to LIFR and GP130 on the surface of mouse cells to activate the downstream signal. Therefore, it is speculated that the human LIF protein secreted by CT26 that highly expresses human LIF can inhibit the immune system of mice. The addition of anti-LIF protein antibodies can relieve this inhibitory effect and thus exert an anti-tumor effect.

[0564] 11.1 Establishment of CT26 cell line overexpressing human LIF gene

[0565] The constructed lentivirus containing the human LIF gene was used to infect the mouse colon cancer cell line CT26. The expression of LIF protein was detected 48 hours after infection. The cell line was cloned by the limiting dilution method, and pressure screening was performed by adding culture medium with a final concentration of 1 μg / mL puromycin. Finally, the CT26 stable cell line CT26-hLIF with high expression of human LIF protein was obtained.

[0566] 11.2 CT26-hLIF BABL / C subcutaneous implant model to detect the anti-tumor activity of anti-human LIF antibody

[0567] CT26-hLIF cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Cells in the logarithmic growth phase were collected and resuspended in PBS to 10 7 After 10 cells / mL, BABL / c mice were inoculated subcutaneously. One day after inoculation, the mice were divided into groups and injected with solvent control and anti-human LIF antibody at a concentration of 15 mg / kg body weight. The drugs were administered twice a week for 4 consecutive weeks. The tumor volume was measured twice a week, the tumor growth curve was drawn, and the tumor inhibition rate was calculated. The results are detailed in Fig.13 The results showed that monoclonal antibody 38E10E1C11 could inhibit the proliferation of CT26-hLIF cells in BABL / c mice.

[0568] 11.3 Testing the sensitivity of different pancreatic cancer cell lines to LIF protein stimulation

[0569] Human pancreatic cancer cell lines Panc02.03, KP4, and MIA paca2 were inoculated into 6-well plates, with 10 6After overnight culture, fresh culture medium was replaced, 50ng / mL recombinant human LIF protein and anti-human LIF antibody 38E10E1C11 were added, and control wells without LIF were set up at the same time. The cells were incubated in a 37°C incubator for 30 minutes, and then the culture medium was removed. 1x cell lysis buffer was added to the cells, 200uL per well, and lysis was performed on ice for 30 minutes. The lysate was transferred to a 1.5mL centrifuge tube, centrifuged at 13,000rpm for 10 minutes, and the supernatant was collected. The supernatant was taken for western blot detection of STAT3 phosphorylation. For details, see Fig.14 The results show that KP4 cell line is most sensitive to human LIF protein stimulation.

[0570] Example 12 Recombinant Expression and Verification of 38E10E1C11 Monoclonal Antibody and P36-033 Monoclonal Antibody

[0571] The light and heavy chain antibody genes of 38E10E1C11 and P36-033 monoclonal antibodies were constructed into the eukaryotic expression vector PCDNA3.1+ using homologous recombination technology, and the recombinant antibodies were expressed using the thermo ExpiCHO expression system. The antibodies were purified using Protein G affinity chromatography, and the purified antibodies were endotoxin-removed using Endotoxin Removal Beads from Changzhou Tiandirenhe Company. The specific experimental methods refer to Example 10.2. Fig.15 The results showed that the 38E10E1C11 antibody (labeled as 38E10E1C11R) recombinantly expressed in CHO cells could inhibit the phosphorylation of STAT3 in KP4 cells induced by human LIF protein.

[0572] Example 13 Identification of the epitope recognized by the 38E10E1C11 antibody

[0573] Preliminary experiments confirmed that the 38E10E1C11 (SEQ ID NOs: 41 and 43) antibody recognizes a linear epitope on the surface of the human LIF protein (SEQ ID NO: 58). This antibody cannot recognize the mouse LIF protein and can block the binding of the human LIF protein to the human LIFR protein. Based on these three points and combined with the analysis of a variety of online protein linear epitope prediction software, it is speculated that the recognition epitope of this antibody is located in the amino acid sequence of 160-202 of the human LIF protein. Therefore, the following hybrid LIF protein was synthesized: Mut3 (SEQ ID NO: 59) replaces the amino acid sequence of the 182-202 position of the human LIF protein with the amino acid sequence of the mouse LIF protein, and Mut4 (SEQ ID NO:60) is to replace the amino acid sequence of the 166-202 position of the human LIF protein with the amino acid sequence of the mouse LIF protein, and the plasmid containing the Mut3, Mut4 hybrid protein and the full-length human LIF protein is transfected into 293T cells. After three days of culture, the cell supernatant is taken for SDS-PAGE electrophoresis and transfer, and the 293T cell culture supernatant is used as a negative control, 38E10E1C11 is used as a primary antibody, and HRP-labeled goat anti-mouse Fab is used as a secondary antibody for detection. At the same time, the M1 cell proliferation experiment is used to detect the activity of the hybrid protein and verify the neutralization activity of 38E10E1C11 on the hybrid protein. At the same time, multiple groups of control wells are set, including control wells with human recombinant LIF protein (rhLIF, purchased from Sino Biological, catalog number: 14890-HNAH), control wells with rhLIF and 38E10E1C11, and control wells without rhLIF and LIF antibody. The results showed that the 38E10E1C11 antibody could recognize denatured full-length LIF protein and Mut3 protein, but could not recognize Mut4 protein ( Fig.16 Sub-figure A in the figure), the M1 cell proliferation experiment showed that the 38E10E1C11 antibody could reverse the inhibition of M1 cell proliferation by full-length LIF protein and Mut3 protein, but could not reverse the inhibition by Mut4 protein ( Fig.16 Sub-figure B), in summary, the recognition epitope of the 38E10E1C11 antibody is located in the amino acids 167-181 of the LIF protein, that is, in the amino acid sequence TYGPDTSGKDVFQKK (SEQ ID NO: 61)

[0574] Example 14 Design, expression and purification of humanized anti-LIF antibody

[0575] The monoclonal antibody 38E10E1C11 obtained from mouse immunization was humanized. Humanization was performed using standard CDR grafting methods. The heavy and light chain regions were cloned from the 38E10E1C11 hybridoma using standard molecular cloning techniques and sequenced using the Sanger method. A BLAST search was then performed on the human heavy and light chain variable sequences, and 3 or 4 sequences were selected from each sequence as humanized receptor frameworks. The heavy chain and light chain CDR1, CDR2 and CDR3 of 38E10E1C11 were cloned into three different heavy chain receptor frameworks (H1-H3) and four different light chain frameworks (L1-L4). At the same time, the HCDR2 of 38E10E1C11 (the amino acid sequence before mutation is shown in SEQ ID NO:45) was subjected to point mutation (the amino acid sequence after mutation is shown in SEQ ID NO:5). The human IgG1 subtype was selected for the heavy chain constant region, and the human Kappa chain was selected for the light chain constant region. The expression vectors containing the humanized antibody heavy chain and humanized antibody light chain genes were co-transfected into 293S cells. The heavy and light chain variable region gene sequences, variable region amino acid sequences, full-length gene sequences and full-length amino acid sequences of the humanized anti-LIF antibodies obtained above are shown in Table 1. Then, the expression levels, antigen binding abilities and thermal stability of 12 different antibody combinations in 293S cells were detected. The 38E10E1C11 chimeric antibody (Chimeric) was used as a positive control. In subsequent experiments, the 38E10E1C11 chimeric antibody was referred to as 38E chimeric antibody or 38E

[0576] Chimeric (SEQ ID NO: 52 and SEQ ID NO: 50). The culture medium was collected, and the expression level of IgG therein was quantified on Gator (similar to Octet), and corrected by ELISA. The antigen binding ability of different combinations was compared by ELISA (Table 2, Table 3).

[0577] Enzyme-linked immunosorbent assay (ELISA):

[0578] Coat each well with 100 μL of 0.5 μg / ml antigen, incubate overnight at 4°C, and wash the plate three times with 300 μL of wash buffer. Block with 200 μL of blocking buffer (2% bovine serum albumin) for 60 minutes at room temperature. Wash the plate three times with 300 μL of wash buffer. Add different concentrations of diluted anti-LIF antibody, 100 μL per well, and incubate at room temperature for 1 hour. Wash the plate 4 times with 300 μL of wash buffer. Add 100 μL of 1:5000 diluted HRP-labeled goat anti-human Fc secondary antibody and incubate at room temperature for 1 hour. Wash the plate 6 times with 300 μL of wash buffer. Add 100 μL of H2O2-Amplx colorimetric solution for color development for 10 minutes at room temperature in the dark. Read the OD 450 value on a microplate reader. Heat treatment: The expression medium was heated at 70°C on a PCR machine for 5 minutes and then quickly cooled to room temperature. Perform subsequent ELISA detection as described above.

[0579] Table 1. Expression levels of different humanized light and heavy chain combinations in 293S cells

[0580]

[0581]

[0582] Table 2. ELISA results of different humanized light and heavy chain combinations expressed in 293S cell supernatants (non-heated)

[0583]

[0584] Table 3. ELISA results of different humanized light and heavy chain combinations in 293S cell expression supernatant (heated)

[0585]

[0586] Example 15 Characterization of selected humanized candidate antibodies using purified IgG samples

[0587] Based on the binding affinity, humanization percentage, antibody expression level and thermal stability data, the present invention selected the following five candidate antibodies for further characterization: H1L1, H1L4, H2L4, H3L2, H3L4, and renumbered these five candidate antibodies as 38E HuH1L1 (SEQ ID NOs: 25 and 9), 38E HuH1L4 (SEQ ID NOs: 25 and 21), 38E HuH2L4 (SEQID NOs: 29 and 21), 38E HuH3L2 (SEQ ID NOs: 33 and 13), 38E HuH3L4 (SEQ ID NOs: 33 and 21), and then co-transfected 293S cells with the selected VH / VL plasmids, harvested the cell culture supernatant, and purified the antibodies by protein A affinity chromatography. Binding ELISA analysis was performed with the purified antibodies to compare the specific binding ability of the humanized antibodies with the 38E chimeric antibodies. The present invention also performed some preliminary analyses to compare their thermal stability and non-specific binding. The results showed that the purified candidate antibodies had very similar antigen binding properties to the 38E chimeric antibody ( Fig.17A , 17C). After treatment at 70°C for 5 minutes, the five humanized antibodies all showed binding abilities similar to those of the chimeric antibody ( Fig. 17B , 17D).

[0588] Example 16 Evaluation of non-specific binding of humanized candidate antibodies

[0589] FACS analysis of LIF-negative HEK293 cells was performed as a preliminary test to assess the potential non-specific binding risk of the antibodies.

[0590] HEK293 cells were trypsinized, washed twice with PBS containing 1% FBS, and resuspended to adjust the cell density to 1.5-2x10 6 / mL, and add the cells to a 96-well U-shaped plate. Adjust the concentration of the antibody to be tested to 20μg / mL, and then perform 3-fold dilutions for a total of 8 concentrations, and set up blank controls and negative controls (Rituxan). Add the diluted antibodies and blank controls to the cells in the 96-well plate, adding 100μL of antibody to each well. Incubate at 4°C for 1 hour, centrifuge at 1000rpm for 5 minutes, carefully discard the supernatant, wash twice with PBS containing 1% FBS, and finally resuspend the cells with 200μL PBS containing 1% FBS for flow cytometric analysis. In the FACS detection of nonspecific binding of HEK293 cells, 38E HuH1L1, 38E HuH3L2, 38EHuH3L4, 38E HuH1L4, 38E chimeric antibodies and negative controls (Rituxan) have similar nonspecific binding affinities to HEK293 cells, while 38E HuH2L4 has higher nonspecific binding to HEK293 cells ( Fig.18 The subgraph A and Fig.18 Subgraph B in .

[0591] Example 17 CE-SDS analysis of antibody purity

[0592] The working concentration for CE-SDS analysis was 1 mg / mL, and the antibody samples were diluted to the specified concentration with loading buffer.

[0593] Preparation of non-reducing CE-SDS electrophoresis samples: Take 95 μL of the diluted sample solution, add 5 μL of 0.8M ammonium iodoacetate aqueous solution, 5 μL of internal reference, and vortex to mix. Take 95 μL of the blank control, add 5 μL of 0.8M ammonium iodoacetate aqueous solution, 5 μL of internal reference, and vortex to mix, which is the non-reducing blank control. Heat at 70°C in a metal bath for 5 minutes, cool to room temperature, and centrifuge at 6000 rpm for 1 minute.

[0594] Preparation of reduction sample solution: Take 95 μL of diluted sample solution, add 5 μL of 2-mercaptoethanol solution and 5 μL of internal reference, and vortex to mix. Take 95 μL of blank control, add 5 μL of 2-mercaptoethanol solution and 5 μL of internal reference, and vortex to mix, which is the reduction blank control.

[0595] Heat in a metal bath at 70°C for 15 minutes, cool to room temperature, and centrifuge at 6000 rpm for 1 minute.

[0596] Sample analysis: 75 μL of sample was added to the test tube, the test tube was placed in the test cup, the test cup was carefully inserted into the sample tray, the test program was run, the injection time of the reduced sample was 30 seconds, the injection time of the non-reduced sample was 40 seconds, the capillary temperature was 20°C, the sample temperature was 20°C, the focusing voltage was 15KV, the focusing time was 40 minutes, and the data was collected using a PDA detector at a wavelength of 214nm. The CE results are shown in Tables 4 and 5.

[0597] Table 4. Reduction CE-SDS results

[0598]

[0599]

[0600] Table 5. Non-reduced CE-SDS results

[0601] Size(KDa) Peak Area(%) Peak ID 38E chimeric 166.39 >99 intact AB 38E HuH1L1 165.93 92.46 intact AB 38E HuH3L2 167.63 93.01 intact AB 38E HuH3L4 166.37 98.82 intact AB

[0602] Example 18 Analysis of Thermal Stability Using Differential Scanning Fluorescence (DSF) / Static Light Scattering (SLS)

[0603] Samples were submitted to the UNcle system (Unchained Labs) for analysis. DSF and SLS were monitored at a temperature ramp of 1°C / min from 25°C to 95°C. UNcle measured SLS at 266nm and 473nm. Tm and Tagg were calculated and analyzed using the UNcle analysis software.

[0604] IgG has multiple domains, each with its own melting temperature (T m ). The CH2 domain typically has a Tm of about 70°C in PBS, and CH3 is more stable with a Tm of about 80°C. Fabs have a wider range of Tms, about 50-85°C, due to their greater sequence variation. Therefore, the Tm values ​​measured by various analytical techniques are usually "apparent" transition temperatures rather than the true Tm values ​​of each domain. Obviously, even this DSF analysis can produce more than one Tm value, and only Tm1 is used to evaluate the thermal stability of therapeutic antibodies. Tagg is the temperature at which SLS begins to detect aggregation. Tagg266 measures SLS at 266nm, which is more sensitive and better suited to detecting smaller aggregated particles. Tagg473 measures SLS at 473nm and is better suited to detecting larger particles.

[0605] As shown in Table 6, the three humanized candidate antibodies all have higher melting temperatures (Tm1) and less aggregation risk than the 38E chimeric antibody.

[0606] Table 6. Differential Scanning Fluorescence (DSF) / Static Light Scattering (SLS) analysis results

[0607]

[0608] Example 19 Analysis of the Aggregation Tendency of Antibodies Using Dynamic Light Scattering (DLS)

[0609] DLS was performed on a UNcle system (Unchained Labs). DLS was measured at 25°C. Data were calculated and analyzed using UNcle analysis software. Dynamic light scattering (DLS) was used to detect aggregation in antibody samples. "Mode diameter" refers to the protein particle diameter, and "mass percentage" refers to the percentage of each particle size fraction. "PDI" is the polydispersity index, and the higher the index, the stronger the polydispersity of the sample. If the PDI is not greater than 0.25, the sample can be considered monodisperse. As shown in Table 7, all 4 antibody samples have a main "peak" (mass fraction exceeds 99%), the PDI of 38E HuH3L4 is better than the chimeric antibody, 38EHuH3L2 is similar to the chimeric antibody, and the PDI of 38E HuH1L1 is worse than the chimeric antibody.

[0610] Table 7. Dynamic light scattering (DLS) analysis results

[0611]

[0612] Example 20 Antibody Affinity Determination

[0613] Gator was used to determine the affinity of anti-LIF antibody to human LIF protein. First, the anti-human LIF antibody was diluted to 5ug / mL with PBS, and then added to the wells AF in the second column of the 96-well plate (200μL per well); the human LIF protein was gradiently diluted with PBS to 100, 50, 25, 12.5, and 6.25μg / mL, respectively, and added to the wells AE in the fourth column of the 96-well plate (100μL per well), and PBS was added to the wells F as a blank control; PBS was added to the wells AF in the first and third columns (200μL per well). The 96-well plate was placed in the instrument and detected with an anti-human Fc biosensor. The experimental results are shown in Table 8, and the results show that the affinity of the three humanized antibodies is close to that of the chimeric antibody.

[0614] Table 8. Humanized anti-LIF antibody affinity determination

[0615] Sample Koff(1 / s) Kon(1 / Ms) KD(M) 38E HuH1L1 4.65E-05 5.69E+05 8.17E-11 38E HuH3L2 3.39E-05 6.31E+05 5.38E-11 38E HuH3L4 4.44E-05 5.79E+05 7.66E-11 38E chimeric 1.57E-05 6.77E+05 2.33E-11

[0616] Example 21 Expression and Purification of Humanized Antibodies and 38E10E1C11 Antibodies

[0617] The variable regions of the light and heavy chains of the humanized antibodies 38E HuH3L2 and 38E HuH3L4 were connected to the mouse antibody constant regions (the heavy chain constant region was mouse IgG1, and the light chain constant region was kappa chain) and cloned into the PCDNA.3.4 vector, respectively, and were named 38EHuH3L2-m (the full-length gene sequences encoding the heavy and light chains of the 38E HuH3L2-m antibody are shown in SEQ ID NO:36 and SEQ ID NO:38, respectively, and the corresponding full-length amino acid sequences of the heavy and light chains of the 38E HuH3L2-m antibody are shown in SEQ ID NO:35 and SEQ ID NO:37, respectively) and 38E HuH3L4-m (the full-length gene sequences encoding the heavy and light chains of the 38E HuH3L4-m antibody are shown in SEQ ID NO:36 and SEQ ID NO:40, respectively, and the corresponding full-length amino acid sequences of the heavy and light chains of the 38E HuH3L4-m antibody are shown in SEQ ID NO:35 and SEQ ID NO:37, respectively). NO:39) used the Expi CHO expression kit of ThermoFisher to perform gene transfection and antibody expression, collected the cell culture supernatant, and purified the antibody using a protein G affinity column. Ultrafiltration tubes were used for ultrafiltration concentration and liquid exchange, and the antibody was finally dissolved in PBS at pH 7.4. The 38E10E1C11 antibody was also expressed and purified in the same way.

[0618] Example 22 Humanized anti-LIF antibody competes with LIFR for binding to human LIF protein

[0619] The recombinant human LIF protein was coated on the ELISA plate at a concentration of 1 μg / mL, and 0.6125 μg / mL of recombinant human LIFR (expressed by fusion with human Fc, purchased from ACRO, catalog number: LIR-H4252) protein was added at 50 μL / well. At the same time, different concentrations of LIF antibodies 38E HuH3L2-m (SEQ ID NOs: 35 and 37), 38E HuH3L4-m (SEQ ID NOs: 35 and 39), 38E10E1C11 (SEQ ID NOs: 41 and 43), P36-033 (SEQ ID NOs: 54 and 55) were added. 56) 100 μL / well, anti-CD3 antibody as negative control (purchased from BioLegend, catalog number: 317326) was incubated at room temperature for 2 hours, washed four times with PBST, and then HRP-labeled goat anti-human Fc antibody was added and incubated at room temperature for 1 hour. After washing four times with PBST, TMB colorimetric solution was added and color was developed at room temperature for 10 minutes. The absorbance value at 450 nm was read by an enzyme reader, and the data were analyzed and plotted using Origin pro 9 software. Fig.19 The results showed that 38E10E1C11, 38E HuH3L2-m, and 38E HuH3L4-m could inhibit the binding of recombinant human LIF to human LIFR. 50 The concentrations of P36-033 and anti-CD3 antibodies were 0.074μg / ml, 0.145μg / ml and 0.103μg / ml respectively. P36-033 had a weak inhibitory effect, and the negative control anti-CD3 antibody could not inhibit the binding of recombinant human LIF to human LIFR.

[0620] Example 23 Humanized anti-LIF antibody does not compete with GP130 for binding to human LIF protein

[0621] The recombinant human LIF protein was coated on the ELISA plate at a concentration of 1 μg / mL, and 12 μg / mL of recombinant human GP130 (expressed by fusion with human Fc, purchased from Sino Biological, Cat. No.: 10974-H03H) protein was added at 50 μL / well. At the same time, different concentrations of LIF antibodies 38E HuH3L2-m ((SEQ ID NOs: 35 and 37), 38E HuH3L4-m (SEQ ID NOs: 35 and 39), P36-033 (SEQ ID NOs: 56 and 54) 100 μL / well, anti-CD28 antibody as negative control (purchased from BioLegend, catalog number: 302914), incubated at room temperature for 2 hours, washed four times with PBST, added HRP-labeled goat anti-human Fc antibody, incubated at room temperature for 1 hour, washed four times with PBST, added TMB colorimetric solution for 10 minutes at room temperature, read the absorbance at 450 nm with an enzyme reader, and analyzed and plotted the data using Origin pro 9 software. Fig. 20 The results showed that humanized antibodies 38EHuH3L2-m, 38E HuH3L4-m and negative control antibody anti-CD28 antibody could not inhibit the binding of recombinant human LIF and human GP130 protein, while P36-033 could inhibit the binding of recombinant human LIF and human GP130 protein.

[0622] Example 24 Detection of Antigen Recognition Specificity of Humanized Anti-LIF Antibody

[0623] Human LIF, human IL-6, human OSM, and human CNTF (all four proteins were purchased from Sino Biological, with catalog numbers: 14890-HNAH; 10395-HNAE; 10452-HNAH; 11841-H07E) were coated on the ELISA plate at a concentration of 1 μg / mL, and different concentrations of LIF antibodies 38E10E1C11, 38E huH3L2-m, and 38E huH3L4-m were added and incubated at room temperature for 1 hour. After washing four times with PBST, HRP-labeled goat anti-mouse Fab secondary antibody was added and incubated at room temperature for 1 hour. After washing four times with PBST, TMB colorimetric solution was added for color development at room temperature for 10 minutes, and the absorbance value at 450 nm was read by an ELISA reader. The data were analyzed and plotted using Origin pro 9 software. For details, see the results. Fig.21 The results showed that 38E10E1C11, 38E huH3L2-m, and 38E huH3L4-m antibodies only bound to human LIF protein but not to human IL-6, OSM, and CNTF.

[0624] Example 25 Identification of epitopes recognized by humanized anti-LIF antibodies

[0625] In the previous experiments, the present invention found that the 38E10E1C11 antibody recognized the linear epitope of the LIF protein. Therefore, the present invention first verified whether the 38E humanized antibody still recognized the linear epitope of the LIF protein. Take the supernatant of 293T cells transfected with human LIF full-length gene sequence, Mut3 and Mut4 protein sequences after 3 days of culture, and the negative control is the supernatant of 293T cell culture, add 5xSDS-PAGE loading buffer, boil for 10 minutes, then take 10μL sample for SDS-PAGE electrophoresis, and then transfer the electrophoresis band to PVDF membrane for western blot detection. The primary antibody used for detection is 38E huH3L2 or 38EhuH3L4 antibody, the antibody concentration is 1μg / mL, incubate at room temperature for 2 hours, wash three times with PBST buffer, add 1:3000 diluted HRP-labeled goat anti-human Fc secondary antibody, incubate at room temperature for 2 hours, wash three times with PBST buffer, add enhanced chemiluminescence solution (Perice, catalog number: 34079) for incubation, and use Amersham Imager 600 ultra-sensitive multifunctional imager for detection and photography. Results are shown in Fig. 22 The results of sub-figure A in the figure show that both humanized antibodies can recognize denatured human LIF protein and Mut3 protein but not Mut4 protein. The same results were obtained in the M1 cell proliferation experiment. Fig. 22 Subgraph B in . Therefore, 38E is determined

[0626] The epitope sequence recognized by huH3L2 and 38E huH3L4 antibodies is TYGPDTSGKDVFQKK (SEQ ID NO: 61).

[0627] Example 26 KP4 cell STAT3 activation inhibition assay to detect the activity of humanized anti-LIF antibody

[0628] After KP4 cells were digested and centrifuged, the cells were resuspended in 1 mL, 5x10 5 cells / well, spread on a 12-well plate. Culture overnight at 37°C, 5% CO2. The next day, discard the original culture medium, add cell culture medium containing 50 ng / mL recombinant human LIF protein and different concentrations of anti-LIF humanized antibodies, set up control wells without recombinant human LIF protein and only with recombinant human LIF protein without antibody, and incubate in a 37°C incubator for 30 minutes. Then remove the culture medium, add 1x cell lysis buffer to the cells, 100 μL per well, and lyse on ice for 30 minutes. Transfer the lysate to a 1.5 mL centrifuge tube, centrifuge at 13,000 rpm for 10 minutes, and collect the supernatant. Take the supernatant for western blot to detect the phosphorylation of STAT3. The results showed that humanized antibodies 38E huH3L4 and 38E huH3L2 can inhibit the phosphorylation of STAT3 induced by LIF protein ( Fig.23 ).

[0629] Example 27 M1 cell proliferation assay to detect the activity of humanized anti-LIF antibody

[0630] After M1 centrifugation, the cells were washed twice with RPMI1640 medium at 2.5 × 10 5 96-well plates were inoculated with a density of 100 μL / mL, 80 μL of cells were inoculated into each well, and culture medium containing 4 ng / mL recombinant human LIF protein and different concentrations of anti-LIF antibodies was added to make the final volume of each well 160 μL. At the same time, control wells without LIF were set up and cultured in a 37°C incubator for 72 hours. CCK-8 was added to detect cell proliferation. The results are detailed in Fig.24 The results showed that humanized antibodies 38E huH3L4-m and 38E huH3L2-m were able to reverse the proliferation inhibition of M1 cells by human LIF protein, with EC50 of 6.52μg / mL and 8.93μg / mL, respectively.

[0631] Example 28: Inhibitory effect of LIF antibody on LIF-induced STAT3 phosphorylation

[0632] 100,000 KP4 cells were seeded in a 96-well plate; LIF antibody diluted in multiple ratios was incubated with 20-100 ng / ml LIF protein at room temperature for 0.5-1 h to form a LIF-Ab mixed solution; the LIF-Ab mixed solution was added to the cell wells and stimulated at 37°C for 5-30 min; the expression levels of P-STAT3 and Total-STAT3 proteins were detected according to the instructions of P-STAT3 (TYR705) KITS (Cisbio) and Total-STAT3 KITS (Cisbio), and the emission signal ratio of the donor and acceptor in each well was calculated: Ratio = Signal 665nm / Signal 620nm

[0633] *10 4 GraphPad Prism software was used to generate data graphs and calculate the inhibition rate of LIF antibody. The results showed that LIF antibody 38EHuH3L4 had an inhibitory effect on LIF-induced STAT3 phosphorylation. Fig.25 As shown, IC 50 It is 3.415nM.

[0634] Example 29: Detection of ADCC activity of LIF antibody

[0635] LIF binds to GP130 and LIFR. Humanized LIF antibody blocks the binding of LIF to LIFR, but cannot block the binding of LIF to GP130. To test whether humanized LIF antibody binds to the cell surface through LIF mediation, thereby causing ADCC. Erbitux antibody ( Merck Serono, positive control) and Human IgG2 (Cat#HG2K, Sino, negative control); then dilute 38EhuH3L4 antibody with ADCC buffer containing LIF protein, three-fold dilution, eight gradients, for standby use; digest DLD-1 cells with trypsin (Cat#25200072, GIBCO), blow off the cells after terminating the reaction and collect them in a centrifuge tube, centrifuge at 1500rpm for 3min, discard the supernatant, resuspend the cells with ADCC buffer and count, adjust the cell concentration, and set aside; resuscitate PBMC cells (Cat#SLB-HP010B, Shanghai Sai Li Biotechnology Co., Ltd.), add 10mL ADCC buffer, centrifuge at 2000rpm for 10min, discard the supernatant, resuspend in ADCC buffer, count, adjust the cell concentration, and set aside; take out the 96-well U-bottom cell culture plate, add 50μl target cell DLD-1, 50μl antibody, and 50μl effector cell PBMC in sequence, the effector target ratio = 30:1, mix well and place at 37℃ 5% The reaction was incubated in a CO2 incubator for 6 h, and LDH was detected using a Cyto Tox96 Non-Radioactive Cytotoxicity Assay Kit (Cat#G1780, Promega), and the absorbance was measured at 490 nm using a microplate reader.

[0636] Calculate the mean absorbance of each parallel well, so that the mean absorbance of all experimental wells, target cell LDH spontaneous release wells (TCR), and effector cell LDH spontaneous release wells (ECR) are subtracted from the mean absorbance of blank medium (CMB). The mean absorbance of target cell LDH maximum release wells (TCM) is subtracted from the mean absorbance of volume correction control wells (VCC). Use the above corrected values ​​to calculate the Cytotoxicity (%) produced by each concentration of antibody according to the following formula:

[0637] Cytotoxicity(%)=(A–B–C) / (D–C)×100%

[0638] A: Average absorbance value after correction of experimental wells

[0639] B: Average absorbance after correction of spontaneous release of LDH from effector cells

[0640] C: Average absorbance after correction of spontaneous release of LDH from target cells

[0641] D: Average absorbance after correction of the maximum release pore of LDH in target cells

[0642] like Fig.26 As shown, the 38E huH3L4 antibody has no ADCC activity.

[0643] References

[0644] 1.Nicola NA,Babon JJ.Leukemia inhibitory factor(LIF).Cytokine GrowthFactor Rev.2015;

[0645] 26(5):533-44.

[0646] 2.Pastuschek J,Poetzsch J,Morales-Prieto DM,Schleuβner E,Markert UR,Georgiev G.Stimulation of the JAK / STAT pathway by LIF and OSM in the humangranulosa cell line COV434.J Reprod Immunol.2015;108:48-55.

[0647] 3.Liu SC,Tsang NM,Chiang WC,Chang KP,Hsueh C,Liang Y,Juang JL,ChowKP,Chang YS.Leukemia inhibitory factor promotes nasopharyngeal carcinomaprogression and radioresistance.J ClinInvest.2013;123(12):5269-83.

[0648] 4.Shi Y,Gao W,Lytle NK,Huang P,Yuan X,Dann AM,et al.Targeting LIF-mediated paracrineinteraction for pancreatic cancer therapy andmonitoring.Nature.2019;569(7754):131-135.

[0649] 5.Cartwright P,McLean C,Sheppard A,RivettD,Jones K,Dalton S.LIF / STAT3controls ES cell self-renewal and pluripotency by a Myc-dependentmechanism.Development 2005;132:885–96.

[0650] 6.Kuphal S,Wallner S,Bosserhoff AK.Impact of LIF(leukemia inhibitoryfactor)expression inmalignant melanoma.Exp Mol Pathol 2013;95:156–65.

[0651] 7.Liu B,Lu Y,Li J,Liu Y,Liu J,WangW.Leukemia inhibitory factorpromotes tumor growth andmetastasis inhuman osteosarcoma via activatingSTAT3.APMIS 2015;123:837–46.

[0652] 8.Morton SD,Cadamuro M,Brivio S,Vismara M,Stecca T,Massani M,etal.Leukemia inhibitoryfactor protects cholangiocarcinoma cells from druginduced apoptosis via a PI3K / AKT-dependent Mcl-1activation.Oncotarget.2015;6:26052–64.

[0653] 9.Kamohara H,Ogawa M,Ishiko T,Sakamoto K,Baba H.Leukemia inhibitoryfactor functions as agrowth factor in pancreas carcinoma cells:involvement ofregulation of LIF and its receptor expression.Int J Oncol.2007;30:977–83.

[0654] 10.Shin JE,Park SH,Jang YK.Epigenetic up-regulation of leukemiainhibitory factor(LIF)gene duringthe progression to breast cancer.Mol Cells2011;31:181–9.

[0655] 11. Li X, Yang Q, Yu H, Wu L, Zhao Y, Zhang C, Yue

[0656] 12. Liu SC, Hsu T, Chang YS, Chung AK, Jiang SS, OuYang CN, Yuh CH, Hsueh C, Liu YP, TsangNM. Cytoplasmic LIF reprograms invasive mode to enhance NPCdissemination through modulating YAP1-FAK / PXN signaling. Nat Commun. 2018; 9(1): 5105.

[0657] 13. Viswanadhapalli S, Luo Y, Sareddy GR, Santhamma B, Zhou M, et al. EC359: A First-in-ClassSmall-Molecule Inhibitor for Targeting Oncogenic LIFRSignaling in Triple-Negative Breast Cancer. Mol Cancer Ther. 2019; 18(8): 1341-1354. Sequence Listing <110> Beijing Jacobs Pharmaceuticals Co., Ltd. <120> Binding molecules specific for LIF and uses thereof <130> JAB-BX300 <150> PCT / CN2019 / 108904 <151> 2019-09-29 <150> PCT / CN2020 / 077049 <151> 2020-02-27 <160> 83 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 1 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 2 Asn Ala Lys Thr Leu Ala Glu 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 3 Gln His His Tyr Val Thr Pro Leu Thr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 4 Ser Tyr Ala Met Ser 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 5 Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Thr Val Lys 1 5 10 15 Gly <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 6 Tyr Tyr Gly Tyr Tyr Phe Asp Phe 1 5 <210> 7 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 7 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 Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 8 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 8 gataccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagccc 120 ggcaagagcc ccaagctgct ggtgtacaac gccagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac ctcggccag 300 ggcaccaagc tggagatcaa gagg 324 <210> 9 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 9 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 Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 10 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 10 gataccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagccc 120 ggcaagagcc ccaagctgct ggtgtacaac gccagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac cttcggccag 300 ggcaccaagc tggagatcaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360 agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642 <210> 11 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 11 Asp Ile His 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 Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Wing Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 12 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 12 gataccaca tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagccc 120 ggcaagagcc ccaagctgct ggtgtacaac gccagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac cttcggccag 300 ggcaccaagc tggagatcaa gagg 324 <210> 13 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 13 Asp Ile His 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 Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 14 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 14 gataccaca tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagccc 120 ggcaagagcc ccaagctgct ggtgtacaac gccaagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac cttcggccag 300 ggcaccaagc tggagatcaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360 agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642 <210> 15 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 15 Asp Ile His Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Has Thr Cys Arg Only Being Glued And Having Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 16 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 16 gataccaca tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagccc 120 ggcaagagcc cccagctgct gtgtacaac gccagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcacccag ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac ctcggccag 300 ggcaccaagc tggagatcaa gagg 324 <210> 17 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 17 Asp Ile His Met Thr Gln Ser Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Has Thr Cys Arg Only Being Glued And Having Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 18 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 18 gacatccaca tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagccc 120 ggcaagagcc cccagctgct ggtgtacaac gccaagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcacccag ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac cttcggccag 300 ggcaccaagc tggagatcaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360 agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642 <210> 19 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 19 Asp Ile His 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 Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 20 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 20 gacatccaca tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagcag 120 ggcaagagcc cccagctgct gtgtacaac gccagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcacccag ttcaccctga agatcacag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac ctcggccag 300 ggcaccaagc tggagatcaa gagg 324 <210> 21 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 21 Asp Ile His Met Thr Gln Ser Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Has Thr Cys Arg Only Being Glued And Having Tyr 20 25 30 Leu Only Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 22 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 22 gacatccaca tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagcag 120 ggcaagagcc cccagctgct ggtgtacaac gccaagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcacccag ttcaccctga agatcaacag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac cttcggccag 300 ggcaccaagc tggagatcaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360 agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642 <210> 23 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 23 Glu Val Met Leu Leu 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 Ile Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Thr Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Thr 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 Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Leu Thr Val Ser Ser 115 <210> 24 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 24 gaggtgatgc tgctggagag cggcggcggc ctggtgcagc cggcggcag cctgaggctg 60 agctgcgccg ccagcggctt catcttcagc agctacgcca tgagctgggt gaggcaggcc 120 cccggcaccg gcctggagtg ggtggccacc atcagcagcg gcggcagcaa cacctacagc 180 cccgacaccg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caggtactac 300 ggctactact tcgacttctg gggccagggc accctgctga ccgtgagcag c 351 <210> 25 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 25 Glu Val Met Leu Leu 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 Ile Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Thr Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Thr 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 Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 26 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 26 gaggtgatgc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt catcttcagc agctacgcca tgagctgggt gaggcaggcc 120 cccggcaccg gcctggagtg ggtggccacc atcagcagcg gcggcagcaa cacctacagc 180 cccgacaccg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caggtactac 300 ggctactact tcgacttctg gggccagggc accctgctga ccgtgagcag cgccagcacc 360 aagggcccca gcgtgttccc cctggcccccc agcagcaaga gcaccagcgg cggcaccgcc 420 gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgag ctggaacagc 480 ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 540 agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgc 600 aacgtgaacc acaagcccag caacaccaag gtggacaaga aggtggagcc caagagctgc 660 gacaagaccc acacctgcccc cccctgcccc gccccccgagc tgctgggcgg ccccagcgtg 720 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc cgaggtgacc 780 tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac 840 ggcgtggagg tgcaacgc caagaccaag cccagggagg agcagtacaa cagcacctac 900 agggtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 960 tgcaaggtga gcaacaagc cctgcccgcc cccatcgaga agaccatcag caaggccaag 1020 ggccagccca gggagcccca ggtgtacacc ctgcccccca gcagggagga gatgaccaag 1080 aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 1140 tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 1200 gacggcagct tcttcctgta cagcaagctg accgtggaca agagcaggtg gcagcagggc 1260 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 1320 ctgagcctga gccccggcaa g 1341 <210> 27 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 27 Glu Val Met 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 Ile Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Thr Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Thr 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 Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Leu Thr Val Ser Ser 115 <210> 28 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 28 gaggtgatgc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt catcttcagc agctacgcca tgagctgggt gaggcaggcc 120 cccggcaccg gcctggagtg gtggcacc atcagcagcg gcggcagca cacctacagc 180 cccgacaccg tgaagggcag gttcaccatc agcagggaca acagcagga caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accccatgt actactgcgc caggtactac 300 ggtactact tcgactctg gggccagggc accctgctga ccgtgagcag c 351 <210> 29 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 29 Glu Val Met 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 Ile Phe Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Thr Gly Leu Glu Trp Val 35 40 45 Only Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Arg Asp Asn Serves Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385,390,395,400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 30 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 30 gaggtgatgc tggtggagag cggcggcggc ctggtgcagc cggcggcag cctgaggctg 60 agctgcgccg ccagcggctt catcttcagc agctacgcca tgagctgggt gaggcaggcc 120 cccggcaccg gcctggagtg ggtggccacc atcagcagcg gcggcagcaa cacctacagc 180 cccgacaccg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccatgt actactgcgc caggtactac 300 ggctact tcgacttctg gggccagggc accctgctga ccgtgagcag cgccagcacc 360 aagggcccca gcgtgttccc cctggcccccc agcagcaaga gcaccagcgg cggcaccgcc 420 gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgag ctggaacagc 480 ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 540 agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgc 600 aacgtgaacc acaagcccag caacaccaag gtggacaaga aggtggagcc caagagctgc 660 gacaagaccc acacctgcccc cccctgcccc gccccccgagc tgctgggcgg ccccagcgtg 720 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc cgaggtgacc 780 tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac 840 ggcgtggagg tgcacaacgc caagaccaag cccagggagg agcagtacaa cagcacctac 900 agggtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggatcaag 960 1020 ggccagccca gggagcccca ggtgtacacc ctgcccccca gcagggagga gatgaccaag 1080 aaccaggtga gcctgacctg cctggtgaag ggctctacc ccagcgacat cgccgtggag 1140 tgggagagca acggccagcc cgagacaac tacaagacca cccccccgt gctggacacc 1200 gacggcagct tctcctgta cagcaagctg accgtggaca agagcaggtg gcagcaggc 1260 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactac ccagaagagc 1320 ctgagcctga gccccggcaa g 1341 <210> 31 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 31 Glu Val Met 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 Ile Phe Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Only Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Arg Asp Asn Serves Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln with Asn Ser Leu Arg with Glu Asp Thr with Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Leu Thr Val Ser Ser 115 <210> 32 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 32 gaggtgatgc tggtggag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt catctcagc agctacgcca tgagctggt gaggcaggcc 120 cccgagacca ggctggagtg ggtggccacc atcagcagcg gcggcagca cacctacagc 180 cccgacaccg tgaagggcag gttcaccatc agcagggaca acagcagga caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accccatgt actactgcgc caggtactac 300 ggtactact tcgactctg gggccagggc accctgctga ccgtgagcag c 351 <210> 33 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 33 Glu Val Met 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 Ile Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Thr 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 Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 34 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 34 gaggtgatgc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt catcttcagc agctacgcca tgagctgggt gaggcaggcc 120 cccgagacca ggctggagtg ggtggccacc atcagcagcg gcggcagcaa cacctacagc 180 cccgacaccg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccatgt actactgcgc caggtactac 300 ggctactact tcgacttctg gggccagggc accctgctga ccgtgagcag cgccagcacc 360 aagggcccca gcgtgttccc cctggccccc agcagcaaga gcaccagcgg cggcaccgcc 420 gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgag ctggaacagc 480 ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 540 agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgc 600 aacgtgaacc acaagcccag caacaccaag gtggacaaga aggtggagcc caagagctgc 660 gacaagaccc acacctgcccc cccctgcccc gccccccgagc tgctgggcgg ccccagcgtg 720 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc cgaggtgacc 780 tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac 840 ggcgtggagg tgcacaacgc caagaccaag cccagggagg agcagtacaa cagcacctac 900 agggtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggatcaag 960 1020 ggccagccca gggagcccca ggtgtacacc ctgcccccca gcagggagga gatgaccaag 1080 aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 1140 tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 1200 gacggcagct tcttcctgta cagcaagctg accgtggaca agagcaggtg gcagcagggc 1260 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 1320 ctgagcctga gccccggcaa g 1341 <210> 35 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 35 Glu Val Met 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 Ile Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Thr 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 Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110 Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys 130 135 140 Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser 145 150 155 160 Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp 180 185 190 Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr 195 200 205 Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys 210 215 220 Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val 245 250 255 Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe 260 265 270 Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu 275 280 285 Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala 305 310 315 320 Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg 325 330 335 Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met 340 345 350 Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro 355 360 365 Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn 370 375 380 Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val 385 390 395 400 Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr 405 410 415 Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu 420 425 430 Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 36 <211> 1323 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 36 gaggtgatgc tggtggagag cggcggcggc ctggtgcagc cggcggcag cctgaggctg 60 agctgcgccg ccagcggctt catcttcagc agctacgcca tgagctgggt gaggcaggcc 120 cccgagacca ggctggagtg ggtggccacc atcagcagcg gcggcagcaa cacctacagc 180 cccgacaccg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag ggccgaggac accgccatgt actactgcgc caggtactac 300 ggctactact tcgacttctg gggccagggc accctgctga ccgtgagcag cgccaagacc 360 acccccca gcgtgtaccc cctggccccc ggcagcgccg cccagaccaa cagcatggtg 420 accctgggct gcctggtgaa gggctacttc cccgagcccg tgaccgtgac ctggaacagc 480 ggcagcctga gcagcggcgt gcacaccttc cccgccgtgc tgcagagcga cctgtacacc 540 ctgagcagca gcgtgaccgt gcccagcagc acctggccca gcgagaccgt gacctgcaac 600 gtggcccacc ccgccagcag caccaaggtg tcgtgcccag ggactgcggc 660 tgcaagccct gcatctgcac cgtgcccgag gtgagcagcg tgttcatctt cccccccaag 720 cccaagggacg tgctgaccat caccctgacc cccaaggtga cctgcgtggt ggtggacatc 780 agcaagggacg accccgaggt gcagttcagc tggttcgtgg agacgtgga ggtgcacacc 840 gcccagaccc agcccaggga ggagcagttc aacagcacct tcaggagcgt gagcgagctg 900 cccatcatgc accaggactg gctgaacggc aaggagttca agtgcagggt gaacagcgcc 960 gccttccccg cccccatcga gaagaccatc agcaagacca agggcaggcc caagccccc 1020 caggtgtaca ccatcccccc ccccaaggag cagatggcca aggacaaggt gagcctgacc 1080 tgcatgatca ccgacttctt ccccgaggac atcaccgtgg agtggcagtg gaacggccag 1140 cccgccgaga actacaagaa cacccagccc atcatggaca ccgacggcag ctacttcgtg 1200 tacagcaagc tgaacgtgca gagagcaac tgggaggccg gcacacctt cacctgcagc 1260 gtgctgcacg agggcctgca caccaccac accgagaga gcctgagcca cagccccggc 1320 Aug 1323 <210> 37 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 37 Asp Ile His Met Thr Gln Ser Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Has Thr Cys Arg Only Being Glued And Having Tyr 20 25 30 Leu Wing Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 38 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 38 gacatccaca tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagccc 120 ggcaagagcc ccaagctgct ggtgtacaac gccaagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac cttcggccag 300 ggcaccaagc tggagatcaa gagggccgac gccgccccca ccgtgagcat cttccccccc 360 agcagcgagc agctgaccag cggcggcgcc agcgtggtgt gcttcctgaa caacttctac 420 cccaaggaca tcaacgtgaa gtggaagatc gacggcagcg agaggcagaa cggcgtgctg 480 aacagctgga ccgaccagga cagcaaggac agcacctaca gcatgagcag caccctgacc 540 ctgaccaagg acgagtacga gaggcacaac agctacacct gcgaggccac ccacaagacc 600 agcaccagcc ccatcgtgaa gagcttcaac aggaacgagt gc 642 <210> 39 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 39 Asp Ile His 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 Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Serves Trp Thr Asp Gln Asp Serves Lys Asp Serves Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 40 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 40 gataccaca tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagcag 120 ggcaagagcc cccagctgct gtgtacaac gccagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcacccag ttcaccctga agatcacag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcac cactacgtga cccccctgac cttcggccag 300 ggcaccaagc tggagatcaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360 agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642 <210> 41 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 41 Asp Ile His Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala For Glu Asn Ile Tyr For Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 42 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 42 gacatccaca tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga gaatatttac agttatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 180 aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat cattatgtta ctccgctcac gttcggtgct 300 gggaccaagc tggagctgaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 360 tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480 aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540 ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600 tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gt 642 <210> 43 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 43 Glu Val Met 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 Ser Gly Phe Ile Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ser Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Ser Val 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 Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys 130 135 140 Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser 145 150 155 160 Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp 180 185 190 Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr 195 200 205 Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys 210 215 220 Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val 245 250 255 Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe 260 265 270 Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu 275 280 285 Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala 305 310 315 320 Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg 325 330 335 Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met 340 345 350 Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro 355 360 365 Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn 370 375 380 Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val 385 390 395 400 Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr 405 410 415 Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu 420 425 430 Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 44 <211> 1323 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 44 gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cattttcagt agttatgcca tgtcttgggt tcgccagagt 120 ccggagacga ggctggagtg ggtcgcaacc attagtagtg gtggtagtaa cacctactct 180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagatattat 300 ggttactact ttgacttctg gggccaaggc accactctca cagtctcctc agccaaaacg 360 acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 420 accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 480 ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 540 ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 600 gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 660 tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 720 cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 780 agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 840 gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 900 cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca 960 gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1020 caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 1080 tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 1140 ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc 1200 tacagcaagc tcaatgtgca gagagcaac tgggaggcag gaatacttt cacctgctct 1260 gtgttacatg agggcctgca caccaccat actgagaga gcctctccca ctctcctggt 1320 aaa 1323 <210> 45 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 45 Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 46 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 46 Asp Ile His Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr With Thr Cys Arg Only Serves As Glu Asn With Tyr 20 25 30 Leu Only Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Serve Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 47 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 47 gataccaca tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga gatatttac agttatttag catggtatca gcagaacag 120 ggaaaatctc ctcagctcct gtctataatg caaaaacctt agcagaaggt gtgccatca 180 ggttcagtgg cagtgatca ggcacatt ttctctgaag atcacagcc tgcagcctga 240 agattttggg agttattact gtcacatca tattgttact cgctcacgt tcggtgctgg 300 gaccaagctg gaggctgaac gggc 324 <210> 48 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 48 Glu Val Met 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 Ser Gly Phe Ile Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ser Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Ser Val 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 Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 49 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 49 gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cattttcagt agttatgcca tgtcttgggt tcgccagagt 120 ccggagacga ggctggagtg ggtcgcaacc attagtagtg gtggtagtaa cacctactct 180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagatattat 300 ggttactact ttgacttctg gggccaaggc accactctca cagtctcctc a 351 <210> 50 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 50 Asp Ile His Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 51 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 51 gacatccaca tgacccagag ccccgccagc ctgagcgcca gcgtgggcga gaccgtgacc 60 atcacctgca gggccagcga gaacatctac agctacctgg cctggtacca gcagaagcag 120 ggcaagagcc cccagctgct ggtgtacaac gccaagaccc tggccgaggg cgtgcccagc 180 aggttcagcg gcagcggcag cggcacccag ttcagcctga agatcaacag cctgcagccc 240 gaggacttcg gcagctacta ctgccagcac cactacgtga cccccctgac cttcggcgcc 300 ggcaccaagc tggagctgaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360 agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480 gagagcgtga ccgagcagga cagcaggac agcacctaca gcctgagcag caccctgacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600 ctgagcagcc ccgtgaccaa gagctcac agggcgagt gc 642 <210> 52 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 52 Glu Val Met 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 Ser Gly Phe Ile Phe Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ser Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Only Thr Ile Will Be Gly Gly Be Asn Thr Tyr Be Pro Asp Be Val 50 55 60 Lys Gly Arg Phe Thr Ile Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 53 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 53 gaggtgatgc tggtggagag cggcggcggc ctggtgaagc ccggcggcag cctgaagctg 60 agctgcgccg ccagcggctt catcttcagc agctacgcca tgagctgggt gaggcagagc 120 cccgagacca ggctggagtg ggtggccacc atcagcagcg gcggcagcaa cacctacagc 180 cccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccctgtac 240 ctgcagatga gcagcctgag gagcgaggac accgccatgt actactgcgc caggtactac 300 ggctactact tcgacttg gggccagggc accaccctga ccgtgagcag cgccagcacc 360 aagggcccca gcgtgttccc cctggccccc agcagcaaga gcaccagcgg cggcaccgcc 420 gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgag ctggaacagc 480 ggcgccctga ccagcggcgt gcacaccttc cccgccgtgc tgcagagcag cggcctgtac 540 agcctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgc 600 aacgtgaacc acaagcccag caacaccaag gtggacaaga aggtggagcc caagagctgc 660 gacaagaccc acacctgcccc cccctgcccc gccccccgagc tgctgggcgg ccccagcgtg 720 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc cgaggtgacc 780 tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac 840 ggcgtggagg tgcacaacgc caagaccaag cccagggagg agcagtacaa cagcacctac 900 agggtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggatcaag 960 1020 ggccagccca gggagcccca ggtgtacacc ctgcccccca gcagggagga gatgaccaag 1080 aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 1140 tgggagagca acggccagcc cgagacaac tacaagacca cccccccgt gctggacacc 1200 gacggcagct tctcctgta cagcaagctg accgtggaca agagcaggtg gcagcaggc 1260 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactac ccagaagagc 1320 ctgagcctga gccccggcaa g 1341 <210> 54 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 54 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Will Have A Thr Cys Lys Flexin, Gln Asp Will Be A N Fluent 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Phe Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Arg Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 55 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 55 gacatcgtga tgacccagtc ccacaagttc atgagcacca gcgtgggcga tcgggtgtcc 60 atcacctgta aggcctccca ggacgtgagc aacgccgtgg cctggtatca gcagaagcct 120 ggccagtccc ctcggctgct gatctattgg gcttccttca ggcacaccgg cgtgcccgat 180 cggttcaccg gctccggatc cggcaccgag tataccctga ccatctcccg ggtgcaggcc 240 gaggatctgg ctctgtatta ttgtcagcag cactacaata ccccctacac cttcggcggc 300 ggcaccaggc tggagatcaa gagagctgat gctgccccca ccgtgagcat cttccctccc 360 tccagcgagc agctgacctc cggcggagcc tccgtggtgt gcttcctgaa caacttctac 420 cccaaggata tcaacgtgaa gtggaagatc gacggcagcg agcggcagaa tggcgtgctg 480 aactcctgga ccgaccagga cagcaaggac tccacctatt ccatgtcctc caccctgacc 540 ctgaccaagg atgagtacga gcggcacaac agctatacct gtgaggccac ccacaagacc 600 tccacctccc ccatcgtgaa gtccttcaat aggaatgagt gc 642 <210> 56 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 56 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Thr Pro Glu Lys Arg Met Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn Gly Gly Asp Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Val Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Val Arg Pro Ser Ala Arg Tyr Asp Glu Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val 115 120 125 Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser 180 185 190 Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala 195 200 205 Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys 210 215 220 Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val 245 250 255 Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe 260 265 270 Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro 290 295 300 Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val 305 310 315 320 Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr 325 330 335 Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys 340 345 350 Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp 355 360 365 Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro 370 375 380 Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser 385 390 395 400 Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala 405 410 415 Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His 420 425 430 His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 57 <211> 1332 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 57 gaggtgatgc tggtggagag cggcggcggc ctggtgcagc ccggcggcag caggaggctg 60 agctgcgccg ccagcggctt caccttcagc agctacccca tgttctgggt gaggcagacc 120 cccgagaaga ggatggagtg ggtggcctac atcagcaacg gcggcgacag cacctactac 180 cccgacaccg tgaagggcag gttcaccgtg agcagggaca acgccaagaa caccctgtac 240 ctgcagatga gcagcctgaa gagcgtggac accgccatct actactgcgt gaggcccagc 300 gccaggtacg acgagtggtt cgcctactgg ggccagggca ccctggtgac cgtgagcagc 360 gccaagacca ccccccccag cgtgtacccc ctggcccccg gcagcgccgc ccagaccaac 420 agcatggtga ccctgggctg cctggtgaag ggctacttcc ccgagcccgt gaccgtgacc 480 tggaacagcg gcagcctgag cagcggcgtg cacaccttcc ccgccgtgct gcagagcgac 540 ctgtacaccc tgagcagcag cgtgaccgtg cccagcagca cctggcccag cgagaccgtg 600 acctgcaacg tggcccaccc cgccagcagc accaaggtgg acaagaagat cgtgcccagg 660 gactgcggct gcaagccctg catctgcacc gtgcccgagg tgagcagcgt gttcatcttc 720 ccccccaagc ccaaggacgt gctgaccatc accctgaccc ccaaggtgac ctgcgtggtg 780 gtggacatca gcaaggacga ccccgaggtg cagttcagct ggttcgtgga cgacgtggag 840 gtgcacaccg cccagaccca gcccagggag gagcagttca acagcacctt caggagcgtg 900 agcgagctgc ccatcatgca ccaggactgg ctgaacggca aggagttcaa gtgcagggtg 960 aacagcgccg ccttccccgc ccccatcgag aagaccatca gcaagaccaa gggcaggccc 1020 aaggcccccc aggtgtacac catccccccc cccaaggagc agatggccaa ggacaaggtg 1080 agcctgacct gcatgatcac cgacttcttc cccgaggaca tcaccgtgga gtggcagtgg 1140 aacggccagc ccgccgagaa ctacaagaac acccagccca tcatggacac cgacggcagc 1200 tacttcgtgt acagcaagct gaacgtgcag aagagcaact gggaggccgg caacaccttc 1260 acctgcagcg tgctgcacga gggcctgcac aaccaccaca ccgagaagag cctgagccac 1320 agccccggca ag 1332 <210> 58 <211> 202 <212> PRT <213> Homo sapiens <400> 58 Met Lys Val Leu Ala Ala Gly Val Val Pro Leu Leu Leu Val Leu His 1 5 10 15 Trp Lys His Gly Ala Gly Ser Pro Leu Pro Ile Thr Pro Val Asn Ala 20 25 30 Thr Cys Ala Ile Arg His Pro Cys His Asn Asn Leu Met Asn Gln Ile 35 40 45 Arg Ser Gln Leu Ala Gln Leu Asn Gly Ser Ala Asn Ala Leu Phe Ile 50 55 60 Leu Tyr Tyr Thr Ala Gln Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys 65 70 75 80 Leu Cys Gly Pro Asn Val Thr Asp Phe Pro Pro Phe His Ala Asn Gly 85 90 95 Thr Glu Lys Ala Lys Leu Val Glu Leu Tyr Arg Ile Val Val Tyr Leu 100 105 110 Gly Thr Ser Leu Gly Asn Ile Thr Arg Asp Gln Lys Ile Leu Asn Pro 115 120 125 Ser Ala Leu Ser Leu His Ser Lys Leu Asn Ala Thr Ala Asp Ile Leu 130 135 140 Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Ser Lys Tyr His 145 150 155 160 Val Gly His Val Asp Val Thr Tyr Gly Pro Asp Thr Ser Gly Lys Asp 165 170 175 Val Phe Gln Lys Lys Lys Leu Gly Cys Gln Leu Leu Gly Lys Tyr Lys 180 185 190 Gln Ile Ile Ala Val Leu Ala Gln Ala Phe 195 200 <210> 59 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 59 Met Lys Val Leu Ala Ala Gly Val Val Pro Leu Leu Leu Val Leu His 1 5 10 15 Trp Lys His Gly Ala Gly Ser Pro Leu Pro Ile Thr Pro Val Asn Ala 20 25 30 Thr Cys Ala Ile Arg His Pro Cys His Asn Asn Leu Met Asn Gln Ile 35 40 45 Arg Ser Gln Leu Ala Gln Leu Asn Gly Ser Ala Asn Ala Leu Phe Ile 50 55 60 Leu Tyr Tyr Thr Ala Gln Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys 65 70 75 80 Leu Cys Gly Pro Asn Val Thr Asp Phe Pro Pro Phe His Ala Asn Gly 85 90 95 Thr Glu Lys Ala Lys Leu Val Glu Leu Tyr Arg Ile Val Val Tyr Leu 100 105 110 Gly Thr Ser Leu Gly Asn Ile Thr Arg Asp Gln Lys Ile Leu Asn Pro 115 120 125 Ser Ala Leu Ser Leu His Ser Lys Leu Asn Ala Thr Ala Asp Ile Leu 130 135 140 Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Ser Lys Tyr His 145 150 155 160 Val Gly His Val Asp Val Thr Tyr Gly Pro Asp Thr Ser Gly Lys Asp 165 170 175 Val Phe Gln Lys Lys Lys Leu Gly Cys Gln Leu Leu Gly Thr Tyr Lys 180 185 190 Gln Val Ile Ser Val Val Val Gln Ala Phe 195 200 <210> 60 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 60 Met Lys Val Leu Ala Ala Gly Val Val Pro Leu Leu Leu Val Leu His 1 5 10 15 Trp Lys His Gly Ala Gly Ser Pro Leu Pro Ile Thr Pro Val Asn Ala 20 25 30 Thr Cys Ala Ile Arg His Pro Cys His Asn Asn Leu Met Asn Gln Ile 35 40 45 Arg Ser Gln Leu Ala Gln Leu Asn Gly Ser Ala Asn Ala Leu Phe Ile 50 55 60 Leu Tyr Tyr Thr Ala Gln Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys 65 70 75 80 Leu Cys Gly Pro Asn Val Thr Asp Phe Pro Pro Phe His Ala Asn Gly 85 90 95 Thr Glu Lys Ala Lys Leu Val Glu Leu Tyr Arg Ile Val Val Tyr Leu 100 105 110 Gly Thr Ser Leu Gly Asn Ile Thr Arg Asp Gln Lys Ile Leu Asn Pro 115 120 125 Ser Ala Leu Ser Leu His Ser Lys Leu Asn Ala Thr Ala Asp Ile Leu 130 135 140 Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Ser Lys Tyr His 145 150 155 160 Val Gly His Val Asp Val Pro Pro Val Pro Asp His Ser Asp Lys Glu 165 170 175 Ala Phe Gln Arg Lys Lys Leu Gly Cys Gln Leu Leu Gly Thr Tyr Lys 180 185 190 Gln Val Ile Ser Val Val Val Gln Ala Phe 195 200 <210> 61 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 61 Thr Tyr Gly Pro Asp Thr Ser Gly Lys Asp Val Phe Gln Lys Lys 1 5 10 15 <210> 62 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 62 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Ala 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Gln Ile Lys Ala Lys Ser Asp Asp Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Cys Trp Glu Trp Asp Leu Asp Phe Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 63 <211> 1344 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 63 caggtgcagc tgcaggag cggcggcggc ctggtgaagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc cacgcctgga tgcactggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgggccag atcaaggcca agagcgacga ctacgccacc 180 tactacgccg agagcgtgaa gggcaggttc accatcagca gggacgacag caagaacacc 240 ctgtacctgc agatgaacag cctgaagacc gaggacaccg ccgtgtacta ctgcacctgc 300 tgggagtggg acctggactt ctggggccag ggcaccatgg tgaccgtgag cagcgccagc 360 accaagggcc ccagcgtgtt ccccctggcc cccagcagca agagcaccag cggcggcacc 420 gccgccctgg gctgcctggt gaaggactac ttccccgagc ccgtgaccgt gagctggaac 480 agcggcgccc tgaccagcgg cgtgcacacc ttccccgccg tgctgcagag cagcggcctg 540 tacagcctga gcagcgtggt gaccgtgccc agcagcagcc tgggcaccca gacctacatc 600 tgcaacgtga accacaagcc cagcaacacc aaggtggaca agagggtgga gcccaagagc 660 tgcgacaaga cccacacctg ccccccctgc cccgcccccg agctgctggg cggccccagc 720 gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagcaggac ccccgaggtg 780 acctgcgtgg tggtggacgt gagccacgag gaccccgagg tgaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcacaa cgccaagacc aagcccaggg aggagcagta caacagcacc 900 tacagggtgg tgagcgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtac 960 aagtgcaagg tgagcaacaa ggccctgccc gcccccatcg agaagaccat cagcaaggcc 1020 aagggccagc ccagggagcc ccaggtgtac accctgcccc ccagcaggga ggagatgacc 1080 aagaaccagg tgagcctgac ctgcctggtg aagggcttct accccagcga catcgccgtg 1140 gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc cgtgctggac 1200 agcgacggca gcttcttcct gtacagcaag ctgaccgtgg acaagagcag gtggcagcag 1260 ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320 agcctgagcc tgagccccgg caag 1344 <210> 64 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 64 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly His Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Ser Val Ser Asn Leu Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala 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 Met Gln Ala 85 90 95 Thr His Ala Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 <h2 style=";text-align:left;direction:ltr">Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 180 185 190<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 195 200 205<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 210 215 220<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 65<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> synthetic polynucleotide<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 65<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gacatcgtga tgacccagac ccccctgagc agccccgtga ccctggggcca gcccgccagc 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atcagctgca ggagcagcca gagcctgctg gacagcgacg gccacaccta cctgaactgg 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgcagcaga ggcccggcca gccccccagg ctgctgatct acagcgtgag caacctggag 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcggcgtgc ccgacaggtt cagcggcagc ggcgccggca ccgacttcac cctgaagatc 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcagggtgg aggccgagga cgtgggcgtg tactactgca tgcaggccac ccacgccccc 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccctacacct tcggccaggg caccaagctg gagatcaaga ggaccgtggc cgcccccagc 360<h2 style=";text-align:left;direction:ltr"> gtgttcatct tccccccag cgacgagcag ctgaagagcg gcaccgccag cgtggtgtgc 420 ctgctgaaca acttctaccc cagggaggcc aaggtgcagt ggaaggtgga caacgccctg 480 cagagcggca acagccagga gagcgtgacc gagcaggaca gcaaggacag cacctacagc 540 ctgagcagca ccctgaccct gagcaaggcc gactacgaga agcacaaggt gtacgcctgc 600 gaggtgaccc accagggcct gagcagcccc gtgaccaaga gcttcaacag gggcgagtgc 660 <210> 66 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 66 Lys Ala Ser Gln Asp Val Ser Donkey Ala Val Ala 1 5 10 <210> 67 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 67 Trp Ala Ser Phe Arg His Thr 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 68 Gln Gln His Tyr Asn Thr Pro Tyr Thr 1 5 <210> 69 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 69 Ser Tyr Pro Met Phe 1 5 <210> 70 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 70 Tyr Ile Ser Asn Gly Gly Asp Ser Thr Tyr Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly <210> 71 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 71 Pro Ser Ala Arg Tyr Asp Glu Trp Phe Ala Tyr 1 5 10 <210> 72 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 72 gacatcgtga tgacccagtc ccacaagttc atgagcacca gcgtgggcga tcgggtgtcc 60 atcacctgta aggcctccca ggacgtgagc aacgccgtgg cctggtatca gcagaagcct 120 ggccagtccc ctcggctgct gatctattgg gcttccttca ggcacaccgg cgtgcccgat 180 cggttcaccg gctccggatc cggcaccgag tataccctga ccatctcccg ggtgcaggcc 240 gaggatctgg ctctgtatta ttgtcagcag cactacaata ccccctacac cttcggcggc 300 ggcaccaggc tggagatcaa g 321 <210> 73 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 73 gaggtgatgc tggtggagag cggcggcggc ctggtgcagc ctggaggatc tcggaggctg 60 agctgtgccg ccagcggctt caccttctcc tcctatccca tgttctgggt gaggcagacc 120 cccgagaagc ggatggagtg ggtggcctat atctccaatg gcggcgattc cacctattat 180 cctgacaccg tgaagggccg gttcaccgtg agccggta accccagaa taccctgtac 240 ctgcagatga gcagcctgaa gtccgtggac accgctatct actattgcgt gaggccctcc 300 gctcggtacg acgagtggtt cgcctattgg ggccaggca ccctggtgac agtgagcgct 360 <210> 74 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 74 Asp Ile His Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr With Thr Cys Arg Only Serves As Glu Asn With Tyr 20 25 30 Leu Only Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Serve Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Val Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala 100 105 <210> 75 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 75 Glu Val Met 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 Ser Gly Phe Ile Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ser Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Asn Thr Tyr Ser Pro Asp Ser Val 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 Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Tyr Tyr Phe Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 76 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 76 gacatccaca tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga gaatatttac agttatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 180 aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat cattatgtta ctccgctcac gttcggtgct 300 gggaccaagc tggagctgaa acgggct 327 <210> 77 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 77 gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cattttcagt agttatgcca tgtcttgggt tcgccagagt 120 ccggagacga ggctggagtg ggtcgcaacc attagtagtg gtggtagtaa cacctactct 180 ccagacagtg tgaagggcg atcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc agatattat 300 ggttactact ttgactctg gggccaggc accactca cagtctccctc a 351 <210> 78 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 78 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Ala 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Gln Ile Lys Ala Lys Ser Asp Asp Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Cys Trp Glu Trp Asp Leu Asp Phe Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 <210> 79 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 79 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly His Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Ser Val Ser Asn Leu Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala 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 Met Gln Ala 85 90 95 Thr His Ala Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val 115 <210> 80 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 80 caggtgcagc tgcaggagag cggcggcggc ctggtgaagc ccggcggcag cctgaggctg 60 agctgcgccg ccagcggctt caccttcagc cacgcctgga tgcactgggt gaggcaggcc 120 cccggcaagg gcctggagtg ggtgggccag atcaaggcca agagcgacga ctacgccacc 180 tactacgccg agagcgtgaa gggcaggttc accatcagca gggacgacag caagaacacc 240 ctgtacctgc agatgaacag cctgaagacc gaggacaccg ccgtgtacta ctgcacctgc 300 tgggagtggg acctggactt ctggggccag ggcaccatgg tgaccgtgag cagc 354 <210> 81 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <h2 style=";text-align:left;direction:ltr"><400> 81<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gacatcgtga tgacccagac ccccctgagc agccccgtga ccctggggcca gcccgccagc 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atcagctgca ggagcagcca gagcctgctg gacagcgacg gccacaccta cctgaactgg 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgcagcaga ggcccggcca gccccccagg ctgctgatct acagcgtgag caacctggag 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcggcgtgc ccgacaggtt cagcggcagc ggcgccggca ccgacttcac cctgaagatc 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcagggtgg aggccgagga cgtgggcgtg tactactgca tgcaggccac ccacgccccc 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccctacacct tcggccaggg caccaagctg gagatcaaga ggaccgtg 348<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 82<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 107<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> synthetic polypeptide<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 82<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Asn Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> Tyr Trp Ala Ser Phe Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Arg Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 83 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 83 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Thr Pro Glu Lys Arg Met Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn Gly Gly Asp Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Val Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Val Arg Pro Ser Ala Arg Tyr Asp Glu Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120

Claims

1. An isolated antibody or antigen-binding portion thereof, wherein the isolated antibody or antigen-binding portion thereof binds to LIF protein, wherein: The isolated antibody or antigen-binding portion thereof comprises: 1) (a) LCDR1 of SEQ ID NO: 1, (b) LCDR2 of SEQ ID NO: 2, (c) LCDR3 of SEQ ID NO: 3, (d) HCDR1 of SEQ ID NO: 4, (e) HCDR2 of SEQ ID NO: 5, and (f) HCDR3 of SEQ ID NO: 6; or 2) (a) LCDR1 of SEQ ID NO: 1, (b) LCDR2 of SEQ ID NO: 2, (c) LCDR3 of SEQ ID NO: 3, (d) HCDR1 of SEQ ID NO: 4, (e) HCDR2 of SEQ ID NO: 45, and (f) HCDR3 of SEQ ID NO:

6.

2. The isolated antibody or antigen-binding portion thereof according to claim 1, wherein The isolated antibody or antigen-binding portion thereof comprises: (a) LCDR1 of SEQ ID NO: 1, (b) LCDR2 of SEQ ID NO: 2, (c) LCDR3 of SEQ ID NO: 3, (d) HCDR1 of SEQ ID NO: 4, (e) HCDR2 of SEQ ID NO: 5, and (f) HCDR3 of SEQ ID NO:

6.

3. The isolated antibody or antigen-binding portion thereof according to claim 1, wherein The isolated antibody or antigen-binding portion thereof comprises: (a) LCDR1 of SEQ ID NO: 1, (b) LCDR2 of SEQ ID NO: 2, (c) LCDR3 of SEQ ID NO: 3, (d) HCDR1 of SEQ ID NO: 4, (e) HCDR2 of SEQ ID NO: 45, and (f) HCDR3 of SEQ ID NO:

6.

4. The isolated antibody or antigen-binding portion thereof according to claim 1, wherein The isolated antibody or antigen-binding portion thereof comprises: 1) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 23; 2) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27; 3) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 7; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31; 4) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 23; 5) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27; 6) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 11; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31; 7) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 23; 8) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27; 9) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 15; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31; 10) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 23; 11) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27; 12) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 19; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31; 13) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 46; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 48; or 14) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 74; and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:

75.

5. The isolated antibody or antigen-binding portion thereof according to claim 4, wherein The isolated antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region respectively comprise an amino acid sequence that is at least 90% homologous to the light chain variable region and the heavy chain variable region selected from 1) to 14) of claim 4.

6. The isolated antibody or antigen-binding portion thereof according to claim 4, wherein The isolated antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region respectively comprise an amino acid sequence that is at least 95% homologous to the light chain variable region and the heavy chain variable region selected from 1) to 14) of claim 4.

7. The isolated antibody or antigen-binding portion thereof according to claim 4, wherein The isolated antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region respectively comprise an amino acid sequence that is at least 96% homologous to the light chain variable region and the heavy chain variable region selected from 1) to 14) of claim 4.

8. The isolated antibody or antigen-binding portion thereof according to claim 4, wherein The isolated antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region respectively comprise an amino acid sequence that is at least 97% homologous to the light chain variable region and the heavy chain variable region selected from 1) to 14) of claim 4.

9. The isolated antibody or antigen-binding portion thereof according to claim 4, wherein The isolated antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region respectively comprise an amino acid sequence that is at least 98% homologous to the light chain variable region and the heavy chain variable region selected from 1) to 14) of claim 4.

10. The isolated antibody or antigen-binding portion thereof according to claim 4, wherein The isolated antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region respectively comprise an amino acid sequence that is at least 99% homologous to the light chain variable region and the heavy chain variable region selected from 1) to 14) of claim 4.

11. The isolated antibody or antigen-binding portion thereof according to claim 4, wherein The isolated antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region respectively comprise an amino acid sequence that is 100% homologous to the light chain variable region and the heavy chain variable region selected from 1) to 14) of claim 4.

12. The isolated antibody or antigen-binding portion thereof according to claim 1, wherein The isolated antibody or antigen-binding portion thereof comprises: 1) a light chain comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; 2) a light chain comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; 3) a light chain comprising the amino acid sequence of SEQ ID NO: 9; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; 4) a light chain comprising the amino acid sequence of SEQ ID NO: 13; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; 5) a light chain comprising the amino acid sequence of SEQ ID NO: 13; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; 6) a light chain comprising the amino acid sequence of SEQ ID NO: 13; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; 7) a light chain comprising the amino acid sequence of SEQ ID NO: 17; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; 8) a light chain comprising the amino acid sequence of SEQ ID NO: 17; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; 9) a light chain comprising the amino acid sequence of SEQ ID NO: 17; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; 10) a light chain comprising the amino acid sequence of SEQ ID NO: 21; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; 11) a light chain comprising the amino acid sequence of SEQ ID NO: 21; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; 12) a light chain comprising the amino acid sequence of SEQ ID NO: 21; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; or 13) a light chain comprising the amino acid sequence of SEQ ID NO: 50; and a heavy chain comprising the amino acid sequence of SEQ ID NO:

52.

13. The isolated antibody or antigen-binding portion thereof according to claim 12, wherein The isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein the light chain and the heavy chain respectively comprise an amino acid sequence that is at least 90% homologous to the light chain and the heavy chain selected from 1) to 13) of claim 12.

14. The isolated antibody or antigen-binding portion thereof according to claim 12, wherein The isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein the light chain and the heavy chain respectively comprise an amino acid sequence that is at least 95% homologous to the light chain and the heavy chain selected from 1) to 13) of claim 12.

15. The isolated antibody or antigen-binding portion thereof according to claim 12, wherein The isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein the light chain and the heavy chain respectively comprise an amino acid sequence that is at least 96% homologous to the light chain and the heavy chain selected from 1) to 13) of claim 12.

16. The isolated antibody or antigen-binding portion thereof according to claim 12, wherein The isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein the light chain and the heavy chain respectively comprise an amino acid sequence that is at least 97% homologous to the light chain and the heavy chain selected from 1) to 13) of claim 12.

17. The isolated antibody or antigen-binding portion thereof according to claim 12, wherein The isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein the light chain and the heavy chain respectively comprise an amino acid sequence that is at least 98% homologous to the light chain and the heavy chain selected from 1) to 13) of claim 12.

18. The isolated antibody or antigen-binding portion thereof according to claim 12, wherein The isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein the light chain and the heavy chain respectively comprise an amino acid sequence that is at least 99% homologous to the light chain and the heavy chain selected from 1) to 13) of claim 12.

19. The isolated antibody or antigen-binding portion thereof according to claim 12, wherein The isolated antibody or antigen-binding portion thereof comprises a light chain and a heavy chain, wherein the light chain and the heavy chain respectively comprise an amino acid sequence that is 100% homologous to the light chain and the heavy chain selected from 1) to 13) of claim 12.

20. An isolated antibody or antigen-binding portion thereof, which binds to LIF protein, wherein: It comprises a light chain variable region (VL), whose amino acid sequence is shown in SEQ ID NO:7, and a heavy chain variable region (VH), whose amino acid sequence is shown in SEQ ID NO:

23.

21. An isolated antibody or antigen-binding portion thereof, which binds to LIF protein, wherein: It comprises a light chain variable region (VL), whose amino acid sequence is shown in SEQ ID NO: 11, and a heavy chain variable region (VH), whose amino acid sequence is shown in SEQ ID NO:

31.

22. An isolated antibody or antigen-binding portion thereof, which binds to LIF protein, wherein: It comprises a light chain variable region (VL), whose amino acid sequence is shown in SEQ ID NO: 19, and a heavy chain variable region (VH), whose amino acid sequence is shown in SEQ ID NO:

31.

23. The isolated antibody or antigen-binding portion thereof according to any one of claims 1 to 22, wherein The isolated antibody is IgG.

24. The isolated antibody or antigen-binding portion thereof according to claim 23, wherein The isolated antibody is IgG1, IgG2 or IgG4.

25. The isolated antibody or antigen-binding portion thereof of claim 24, wherein The isolated antibody is a monoclonal antibody, a chimeric antibody, a human antibody, a bispecific antibody, Fab, Fab', Fab'-SH or F(ab')2.

26. The isolated antibody or antigen-binding portion thereof of claim 24, wherein The isolated antibody is a humanized antibody, Fv or single chain antibody (scFv).

27. The isolated antibody or antigen-binding portion thereof of claim 26, wherein The isolated antibody or antigen-binding portion thereof is a LIF (leukemia inhibitory factor) antagonist.

28. The isolated antibody or antigen-binding portion thereof of claim 27, wherein The isolated antibody or antigen-binding portion thereof can inhibit LIF expression and / or block LIF activity.

29. The isolated antibody or antigen-binding portion thereof of claim 28, wherein The isolated antibody or antigen-binding portion thereof is capable of competing or cross-competing for binding to LIF.

30. A nucleotide composition comprising a nucleotide molecule encoding the isolated antibody or antigen binding portion thereof of any one of claims 1-22 or 24-29.

31. The nucleotide composition according to claim 30, comprising: 1) A first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23; 2) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27; 3) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 7; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31; 4) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23; 5) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27; 6) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 11; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31; 7) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23; 8) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27; 9) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 15; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31; 10) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 23; 11) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 27; 12) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 19; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 31; 13) a first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 46; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 48; or 14) A first nucleic acid molecule comprising a DNA encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 74; and a second nucleic acid molecule comprising a DNA encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO:

75.

32. The nucleotide composition according to claim 31, wherein The DNA encoding the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:7 is shown in SEQ ID NO:

8.

33. The nucleotide composition according to claim 31, wherein The DNA encoding the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:11 is shown in SEQ ID NO:

12.

34. The nucleotide composition according to claim 31, wherein The DNA encoding the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:15 is shown in SEQ ID NO:

16.

35. The nucleotide composition according to claim 31, wherein The DNA encoding the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:19 is shown in SEQ ID NO:

20.

36. The nucleotide composition according to claim 31, wherein The DNA encoding the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:46 is shown in SEQ ID NO:

47.

37. The nucleotide composition according to claim 31, wherein The DNA encoding the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO:74 is shown in SEQ ID NO:

76.

38. The nucleotide composition according to claim 31, wherein The DNA encoding the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:23 is shown in SEQ ID NO:

24.

39. The nucleotide composition according to claim 31, wherein The DNA encoding the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:27 is shown in SEQ ID NO:

28.

40. The nucleotide composition according to claim 31, wherein The DNA encoding the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:31 is shown in SEQ ID NO:

32.

41. The nucleotide composition according to claim 31, wherein The DNA encoding the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:48 is shown in SEQ ID NO:

49.

42. The nucleotide composition according to claim 31, wherein The DNA encoding the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO:75 is shown in SEQ ID NO:

77.

43. The nucleotide composition according to claim 30, wherein The nucleotide composition comprises: 1) A first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 9; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25; 2) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 9; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 29; 3) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 9; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 33; 4) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 13; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25; 5) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 13; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 29; 6) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 13; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 33; 7) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25; 8) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 29; 9) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 17; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 33; 10) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 21; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25; 11) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 21; and a second nucleic acid molecule comprising a DNA encoding the heavy chain of the amino acid sequence of SEQ ID NO: 29; 12) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 21; and a second nucleic acid molecule comprising a DNA encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 33; or 13) a first nucleic acid molecule comprising a DNA encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 50; and a second nucleic acid molecule comprising a DNA encoding the heavy chain having an amino acid sequence as shown in SEQ ID NO:

52.

44. The nucleotide composition according to claim 43, wherein The DNA encoding the light chain having the amino acid sequence shown in SEQ ID NO:13 is shown in SEQ ID NO:

14.

45. The nucleotide composition according to claim 43, wherein The DNA encoding the light chain having the amino acid sequence shown in SEQ ID NO:17 is shown in SEQ ID NO:

18.

46. ​​The nucleotide composition according to claim 43, wherein The DNA encoding the light chain having the amino acid sequence shown in SEQ ID NO:21 is shown in SEQ ID NO:

22.

47. The nucleotide composition according to claim 43, wherein The DNA encoding the light chain having the amino acid sequence shown in SEQ ID NO:50 is shown in SEQ ID NO:

51.

48. The nucleotide composition according to claim 43, wherein The DNA encoding the heavy chain having the amino acid sequence shown in SEQ ID NO:25 is shown in SEQ ID NO:

26.

49. The nucleotide composition according to claim 43, wherein The DNA encoding the heavy chain having the amino acid sequence shown in SEQ ID NO:29 is shown in SEQ ID NO:

30.

50. The nucleotide composition according to claim 43, wherein The DNA encoding the heavy chain having the amino acid sequence shown in SEQ ID NO:33 is shown in SEQ ID NO:

34.

51. The nucleotide composition according to claim 43, wherein The DNA encoding the heavy chain having the amino acid sequence shown in SEQ ID NO:52 is shown in SEQ ID NO:

53.

52. A vector comprising the nucleotide composition of any one of claims 31-51.

53. The vector according to claim 52, wherein The vector is a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.

54. A host cell comprising the vector of claim 53.

55. The host cell according to claim 54, wherein The host cell containing the vector is obtained by transformation of the vector.

56. The host cell according to claim 55, wherein The host cell is a bacterial, yeast or mammalian cell.

57. The host cell according to claim 56, wherein The host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells.

58. A method for preparing the isolated antibody or antigen-binding portion thereof of any one of claims 1-22 or 24-29, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell of any one of claims 54-57, and isolating the antibody or antigen-binding fragment thereof.

59. A pharmaceutical composition comprising a therapeutically effective amount of the isolated antibody or antigen-binding portion thereof of any one of claims 1-22 or 24-29, and a pharmaceutically acceptable excipient.

60. A reagent for measuring LIF in a biological sample, comprising the isolated antibody or antigen-binding portion thereof of any one of claims 1-22 or 24-29.

61. The reagent according to claim 60, wherein The biological sample is blood, serum, urine or biopsy material.

62. The reagent according to claim 60, wherein The biological sample is a tumor.

63. The reagent according to claim 60, wherein The biological sample is any tissue suspected of containing abnormal LIF levels.

64. Use of the isolated antibody or antigen-binding portion thereof of any one of claims 1-22 or 24-29 in the preparation of a medicament for treating a disease or condition associated with LIF, wherein: The disease or disorder associated with LIF is a solid tumor, which is colorectal cancer or pancreatic cancer.

65. Use of the pharmaceutical composition of claim 59 in the preparation of a medicament for treating a disease or condition associated with LIF, wherein: The disease or disorder associated with LIF is a solid tumor, and the solid tumor is colorectal cancer or pancreatic cancer.

66. The use according to claim 64 or 65, wherein The drug is used for inhibiting LIF expression and / or blocking LIF activity.

67. The isolated antibody or antigen-binding portion thereof of any one of claims 1-22 or 24-29 for use in treating a disease or condition associated with LIF, wherein: The disease or disorder associated with LIF is a tumor, wherein the tumor is a solid tumor, wherein the solid tumor is colorectal cancer or pancreatic cancer.

68. The pharmaceutical composition of claim 59 for treating a disease or condition associated with LIF, wherein: The disease or disorder associated with LIF is a tumor, wherein the tumor is a solid tumor, wherein the solid tumor is colorectal cancer or pancreatic cancer.

Images