NZ788350A - Binding molecule specific for LIF and use thereof - Google Patents

Binding molecule specific for LIF and use thereof

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Publication number
NZ788350A
NZ788350A NZ788350A NZ78835020A NZ788350A NZ 788350 A NZ788350 A NZ 788350A NZ 788350 A NZ788350 A NZ 788350A NZ 78835020 A NZ78835020 A NZ 78835020A NZ 788350 A NZ788350 A NZ 788350A
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NZ
New Zealand
Prior art keywords
seq
amino acid
acid sequence
represented
variable region
Prior art date
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NZ788350A
Inventor
Shanshan He
Qinghao Liu
Jun Tao
Hongling Wang
Guiqun Yang
Haiyan Yang
Wenlai Zhou
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Jacobio Pharmaceuticals Co Ltd
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Application filed by Jacobio Pharmaceuticals Co Ltd filed Critical Jacobio Pharmaceuticals Co Ltd
Publication of NZ788350A publication Critical patent/NZ788350A/en

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Abstract

Provided is a binding molecule specific for LIF and use thereof. Specifically, provided is an isolated antibody or an antigen-binding fragment thereof that binds to LIF and inhibits the activity of LIF. Also provided are uses of the isolated antibody or the antigen-binding fragment thereof in treatment of diseases. ent of diseases.

Description

The present application is a divisional application from New Zealand Patent Application Number 786895, which is herein incorporated by reference.
DESCRIPTION BINDING MOLECULE SPECIFIC FOR LIF AND USE THEREOF Technical Field The invention s to an isolated dy or antigen-binding fragment thereof specifically binding to LIF, and the use of the isolated antibody or antigen-binding fragment f of the invention, and the treatment method using the isolated antibody or n-binding fragment f of the invention.
Background Art Leukemia inhibitory factor (LIF) is a member of the IL6 type cytokines, and it has various biological activity including stimulating or ting each of cell eration, entiation and survival [1]. Human LIF n has 202 amino acids, and it has two receptors on the cell membrane surface, GP130 and LIFR. The LIF protein binds to these two receptors, causing the two receptors to form a heterodimer, thereby activating the downstream signaling pathways, such as MAPK signaling pathway and JAK/STAT signaling pathway [2]. It has reported that overexpression of LIF protein and increased serum levels of LIF protein are correlated with poor prognosis of multiple tumors [3, 4]. LIF is a key regulator of cancer stem cells, plays an important role in stem cell maintenance, self-renewal and pluripotency, etc., and is associated with chemoresistance [5, 6]. In addition, LIF can also e the growth and metastasis of the tumor [7]. Recent ce indicates that LIF upregulated JAK– STAT3 signaling pathway via autocrine and paracrine mechanisms in tumors, y playing a role of promoting tumor growth and inhibiting immune response [8, 9, 10].
Therefore, LIF is a potential therapeutic target. However, the currently developed treatment method for LIF targets is not optimistic. For example, many literatures report that reducing the expression of LIF protein by RNA interference can inhibit tumor growth [11, 12], but the technique of RNA erence has the weakness of poor targeting, short half-life, poor membrane permeability, and is difficult to make medicine. EC359 is a small molecule inhibitor for LIFR. It can not only inhibit the binding of LIFR to LIF, but also inhibit the binding of OSM, CTF1 and CNTF to LIFR [13]. It is unknown whether these additional inhibitions will lead to additional toxicity, and the small molecule inhibitors specific for LIF protein have not been reported yet. Only one antibody drug targeting LIF protein is currently in clinical development stage, and its relevant safety and efficacy data have not yet been published. ore, more researches are needed to develop drugs and combination therapies for LIF targets.
Summary of ion The invention provides an ed antibody or antigen-binding fragment specifically binding to LIF and the use thereof in the treatment of diseases.
In one respect, the ion provides an isolated dy or antigen-binding fragment thereof that binds at an epitope ented by an amino acid sequence TYGPDTSGKDVFQKK(SEQ ID NO: 61) of human LIF protein or at an epitope of the corresponding amino acid sequence of a ent mammalian species.
In another respect, the invention provides an isolated antibody or antigen-binding fragment thereof, which comprises: (a) LCDR1 sing an amino acid sequence ed from a group consisting of SEQ ID NO: 1 or 66, and conservative modifications thereof; (b) LCDR2 comprising an amino acid sequence selected from a group consisting of SEQ ID NO: 2 or 67, and conservative modifications thereof; (c) LCDR3 comprising an amino acid sequence selected from a group consisting of SEQ ID NO: 3 or 68, and conservative modifications f; (d) HCDR1 comprising an amino acid sequence selected from a group consisting of SEQ ID NO: 4 or 69, and conservative modifications thereof; (e) HCDR2 comprising an amino acid sequence selected from a group consisting of SEQ ID NO: 5 , 45, or 70, and conservative modifications thereof; and (f) HCDR3 comprising an amino acid sequence selected from a group consisting of SEQ ID NO: 6 or 71, and conservative modifications thereof.
Optionally, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 has additions, substitutions, ons and/or insertions of 17 or less amino acids.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) LCDR1 sing an amino acid ce selected from a group consisting of SEQ ID NO: 1, and conservative modifications thereof; (b) LCDR2 comprising an amino acid ce selected from a group ting of SEQ ID NO: 2, and conservative modifications thereof; (c) LCDR3 comprising an amino acid sequence selected from a group consisting of SEQ ID NO: 3, and conservative modifications thereof; (d) HCDR1 comprising an amino acid sequence ed from a group consisting of SEQ ID NO: 4, and conservative modifications thereof; (e) HCDR2 comprising an amino acid sequence selected from a group consisting of SEQ ID NO: 5 or 45, and conservative modifications thereof; (f) HCDR3 comprising an amino acid ce ed from a group consisting of SEQ ID NO: 6, and conservative modifications thereof.
Optionally, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 has additions, substitutions, deletions and/or insertions of 17 or less amino acids.
In some embodiments, optionally, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 has additions, substitutions, deletions and/or insertions of 9 or less amino acids.
In some embodiments, optionally, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 or HCDR3 has additions, substitutions, deletions and/or insertions of 5 or less amino acids.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: 1) (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; 2) (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c) LCDR3 sing 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; or 3) (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.
In some embodiments, the isolated antibody or n-binding fragment thereof comprises: 1) (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 comprising SEQ ID NO: 2, (c) LCDR3 comprising SEQ ID NO: 3, (d) HCDR1 sing SEQ ID NO: 4, (e) HCDR2 comprising SEQ ID NO: 5, and (f) HCDR3 comprising SEQ ID NO: 6; or 2) (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.
In some embodiments, the isolated antibody or antigen-binding fragment f comprises: (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 sing 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.
In some embodiments, the isolated antibody or antigen-binding fragment 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.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (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.
In some embodiments, the isolated antibody or antigen-binding nt f is a murine antibody or n-binding fragment thereof, a ic antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
In some embodiments, the isolated antibody is a humanized antibody comprising a framework region or a framework region variant thereof derived from human antibody.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (i) a light chain variable region (VL) comprising an amino acid sequence with at least 85% identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 7, 11, 15, 19, 46, 74 or 82, and conservative modifications thereof; and (ii) a heavy chain variable region (VH) comprising an amino acid sequence with at least 85% identity to an amino acid sequence ed from a group consisting of SEQ ID NO: 23, 27, 31, 48, 75 or 83, and conservative modifications thereof.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (i) a light chain variable region (VL) comprising an amino acid sequence with at least 85% identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 7, 11, 15, 19 or 46, and conservative cations thereof; and (ii) a heavy chain variable region (VH) comprising an amino acid sequence with at least 85% identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 23, 27, 31 or 48, and conservative modifications thereof.
In some embodiments, the isolated antibody or antigen-binding fragment f comprises: (i) a light chain variable region (VL) comprising an amino acid sequence with at least 85% identity to an amino acid sequence ed from a group consisting of SEQ ID NO: 7, 11, 15 or 19, and vative modifications thereof; and (ii) a heavy chain variable region (VH) comprising an amino acid sequence with at least 85% identity to an amino acid ce selected from a group ting of SEQ ID NO: 23, 27 or 31, and conservative modifications f.
In some embodiments, the light chain variable region ses an amino acid ce with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the light chain variable region selected from (i), and the heavy chain le region comprises an amino acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the heavy chain variable region selected from (ii).
In some embodiments, the ed antibody or antigen-binding fragment thereof comprises: 1) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23; 2) a light chain le region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 27; 3) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain le region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; 4) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23; ) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 27; 6) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; 7)a light chain variable region (VL) that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23; 8)a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 27; 9)a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; ) a light chain variable region (VL) that ses an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23; 11) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 27; 12) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; 13) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 46, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 48; 14) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 74, and a heavy chain le region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 75; or ) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 82, and a heavy chain variable region (VH) that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO:83.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: 1) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 23; 2) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 27; 3) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 31; 4) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23; ) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 11, and a heavy chain le region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 27; 6) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; 7) a light chain variable region (VL) that comprises an amino acid ce with at least 85% ty to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23; 8) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain le region (VH) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 27; 9) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; ) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 23; 11) a light chain variable region (VL) that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 27; or 12) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the isolated antibody or antigen-binding nt thereof comprises: a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23.
In some ments, the ed antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region(VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 27.
In some embodiments, the isolated antibody or antigen-binding nt thereof comprises: a light chain variable region (VL) that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23.
In some embodiments, the isolated antibody or antigen-binding nt thereof comprises: a light chain variable region (VL) that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 27.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain le region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 27.
In some embodiments, the ed dy or antigen-binding fragment thereof comprises: a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain le region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23.
In some ments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain le region (VH) that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 27.
In some embodiments, the isolated antibody or antigen-binding nt thereof comprises: a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that ses an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the isolated antibody or antigen-binding fragment thereof a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 46, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 48.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 74, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 75.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 82, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO:83.
In some embodiments, the light and heavy chain variable region comprise an amino acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the light and heavy chain le region selected from 1)-15), respectively.
In one respect, the invention provides an isolated antibody or antigen-binding fragment thereof comprising a combination of a heavy and a light chain variable region selected from any one of the following (i) to (ii): (i) a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 that have the same ce as any one of SEQ ID NO: 7, 11, 15, 19, 46 or 82; and (ii) a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 that have the same sequence as any one of SEQ ID NO: 23, 27, 31, 48 or 83.
In some embodiments, the ed antibody or antigen-binding fragment thereof comprises a light and a heavy chain, wherein: (I) the light chain comprises an amino acid sequence with at least 85% ty to an amino acid sequence selected from a group consisting of SEQ ID NO: 9, 13, 17, 21, 37, 39, 50 or 54, and conservative modifications f; and (II) the heavy chain comprises an amino acid sequence with at least 85% identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 25, 29, 33, , 52 or 56, and conservative modifications f.
In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a light and a heavy chain, wherein: (I) the light chain comprises an amino acid sequence with at least 85% identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 9, 13, 17, 21, 37, 39 or 50, and conservative modifications thereof; and (II) the heavy chain ses an amino acid ce with at least 85% identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 25, 29, 33, or 52, and conservative modifications thereof.
In some embodiments, the ed antibody or antigen-binding fragment thereof comprises a light and a heavy chain, wherein: (I) the light chain comprises an amino acid sequence with at least 85% identity to an amino acid sequence selected from a group consisting of SEQ ID NO: 9, 13, 17 or 21, and conservative modifications thereof; and (II) the heavy chain comprises an amino acid sequence with at least 85% identity to an amino acid sequence selected from a group ting of SEQ ID NO: 25, 29 or 33, and conservative modifications thereof.
In some embodiments, the light chain comprises an amino acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an light chain selected from (I), and the heavy chain comprises an amino acid ce with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an heavy chain selected from (II).
In some embodiments, the isolated antibody or antigen-binding nt thereof 1) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 2) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 3) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 4) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: ) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 6) a light chain that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 8) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: ) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 21, and a heavy chain that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 25, 11) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 21, and a heavy chain that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 29, 12) a light chain that comprises an amino acid ce with at least 85% ty to an amino acid sequence of SEQ ID NO: 21, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 33, 13) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 37, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 35, 14) a light chain that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 39, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 35, ) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 50, and a heavy chain that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 52, or 16) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 54, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 56.
In some embodiments, the light and heavy chain comprise an amino acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% ty to a light and heavy chain selected from 1)-16) , respectively.
In some embodiments, the isolated antibody or n-binding fragment thereof comprises: a light chain that comprises an amino acid ce with at least 85% ty to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated dy or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment f comprises: a light chain that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: In some embodiments, the ed antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some ments, the isolated antibody or antigen-binding fragment f comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding nt thereof comprises: a light chain that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 21, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 21, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 21, and a heavy chain that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the ed antibody or n-binding fragment thereof comprises: a light chain that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 37, and a heavy chain that ses an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 39, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 50, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 54, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: In another respect, the invention es an isolated antibody or antigen-binding fragment thereof sing (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.
In yet another respect, the invention provides an isolated antibody or n-binding fragment thereof comprising (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 sing SEQ ID NO: 2, (c) LCDR3 comprising SEQ ID NO: 3, (d) HCDR1 comprising SEQ ID NO: 4, (e) HCDR2 sing SEQ ID NO: 45, and (f) HCDR3 comprising SEQ ID NO: 6.
In yet another respect, the invention provides an isolated antibody or n-binding fragment thereof comprising a light chain variable region (VL) represented by SEQ ID NO: 7, and a heavy chain variable region (VH) represented by SEQ ID NO: 23.
In yet another respect, the invention provides an isolated dy or n-binding nt thereof comprising a light chain variable region (VL) ented by SEQ ID NO: 11, and a heavy chain variable region (VH) represented by SEQ ID NO: 31.
In yet another respect, the invention provides an isolated antibody or antigen-binding fragment thereof comprising a light chain le region (VL) represented by SEQ ID NO: 19, and a heavy chain variable region (VH) represented by SEQ ID NO: 31.
In some 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), a Fab, a Fab’, a Fab’-SH or a F(ab’)2.
In some embodiments, the ed antibody is an IgG.
In some embodiments, the isolated antibody is an IgG1, IgG2 or IgG4.
In some embodiments, the isolated antibody or antigen-binding fragment thereof is leukemia inhibitory factor (LIF) antagonist.
In some embodiments, the isolated antibody or antigen-binding fragment thereof is capable of inhibiting the expression of LIF and/or blocking the activity of LIF.
In some embodiments, the ed antibody or antigen-binding fragment thereof is capable of competing or cross competing for binding to LIF.
In yet another respect, the invention provides a nucleotide ition comprising a nucleotide molecule encoding the isolated antibody or antigen-binding fragment thereof of the invention. In some embodiments, the nucleotide molecule is DNA or RNA. In some embodiments, the nucleotide molecule is DNA.
In some embodiments, the nucleotide composition comprises: (i) a first nucleic acid molecule comprising DNA encoding a light chain le region (VL) as represented by an amino acid ce of SEQ ID NO: 7, 11, 15, 19, 46 or 82 ; and (ii) a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23, 27, 31, 48 or 83.
In some embodiments, the nucleotide ition comprises: (i) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7, 11, 15, 19 or 46; and (ii) a second nucleic acid le comprising DNA encoding a heavy chain le region (VH) as represented by an amino acid sequence of SEQ ID NO: 23, 27, 31 or In some embodiments, the nucleotide composition ses: (i) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7, 11, 15, or 19; (ii) a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23, 27 or 31.
In some ments, the first nucleic acid molecule comprises DNA with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the first nucleic acid molecule selected from (i); the second nucleic acid molecule comprises DNA with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the second nucleic acid le selected from (ii).
In some embodiments, the nucleotide composition comprises: 1) a first nucleic acid molecule comprising DNA encoding a light chain le region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second c acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 2) a first nucleic acid molecule sing DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; 3) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid le comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31; 4) a first nucleic acid molecule sing DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; ) a first nucleic acid molecule sing DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; 6) a first nucleic acid le comprising DNA ng a light chain variable region (VL) as represented by an amino acid ce of SEQ ID NO: 11 and a second c acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31; 7) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 8) a first nucleic acid le comprising DNA encoding a light chain variable region (VL) as ented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule sing DNA encoding a heavy chain variable region (VH) as represented by an amino acid ce of SEQ ID NO: 27; 9) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31; ) a first nucleic acid molecule sing DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 11) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; 12) a first c acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31; 13) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 46 and a second nucleic acid molecule comprising DNA encoding a heavy chain le region (VH) as represented by an amino acid sequence of SEQ ID NO: 48; 14) a first nucleic acid molecule comprising DNA ng a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 74 and a second c acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 75; or ) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 82 and a second nucleic acid molecule comprising DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 83.
In some embodiments, the nucleotide composition comprises: 1) a first c acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid ce of SEQ ID NO: 7 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 2) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; 3) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second c acid molecule comprising DNA encoding a heavy chain variable region (VH) as ented by an amino acid sequence of SEQ ID NO: 31; 4) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as ented by an amino acid sequence of SEQ ID NO: 23; ) a first c acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second nucleic acid molecule sing DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; 6) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid ce of SEQ ID NO: 31; 7) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as ented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 8) a first nucleic acid le comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA ng a heavy chain le region (VH) as represented by an amino acid ce of SEQ ID NO: 27; 9) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as ented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule sing DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31; ) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as ented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 11) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; or 12) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the nucleotide ition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid ce of SEQ ID NO: 23.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid molecule sing DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second c acid le comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule sing DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second nucleic acid molecule comprising DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second nucleic acid molecule comprising DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 15 and a second c acid molecule comprising DNA encoding a heavy chain le region (VH) as ented by an amino acid sequence of SEQ ID NO: 23.
In some embodiments, the nucleotide composition comprises: a first nucleic acid le comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule sing DNA encoding a heavy chain variable region (VH) as ented by an amino acid sequence of SEQ ID NO: 23.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain le region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as ented by an amino acid sequence of SEQ ID NO: 27.
In some embodiments, the nucleotide ition comprises: a first nucleic acid le comprising DNA encoding a light chain variable region (VL) as represented by an amino acid ce of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as ented by an amino acid sequence of SEQ ID NO: 31; In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 46 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as ented by an amino acid sequence of SEQ ID NO: 48.
In some embodiments, the nucleotide ition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 74 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 75.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 82 and a second nucleic acid molecule comprising DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 83.
In some ments, the first nucleic acid sequence and the second nucleic acid sequence se a DNA sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the first nucleic acid sequence or the second nucleic acid sequence ed from , respectively.
In some embodiments, DNA ng a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 is shown as SEQ ID NO: 8.
In some embodiments, DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 is shown as SEQ ID NO: In some embodiments, DNA encoding a light chain variable region (VL) as ented by an amino acid sequence of SEQ ID NO: 15 is shown as SEQ ID NO: In some embodiments, DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 is shown as SEQ ID NO: In some embodiments, DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 46 is shown as SEQ ID NO: In some embodiments, DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 74 is shown as SEQ ID NO: In some embodiments, DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 82 is shown as SEQ ID NO: In some embodiments, DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23 is shown as SEQ ID NO: In some ments, DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27 is shown as SEQ ID NO: In some embodiments, DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31 is shown as SEQ ID NO: In some embodiments, DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 48 is shown as SEQ ID NO: In some embodiments, DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 75 is shown as SEQ ID NO: In some embodiments, DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 83 is shown as SEQ ID NO: In some embodiments, the nucleotide composition comprises: (I)a first nucleic acid sequence comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9, 13, 17, 21, 37, 39, 50 or 54; (II) a second nucleic acid sequence comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25, 29, 33, 35, 52 or 56.
In some embodiments, the tide ition comprises: (I) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9, 13, 17, 21, 37, 39 or 50; (II) a second c acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25, 29, 33, 35 or 52.
In some embodiments, the nucleotide composition comprises: (I) a first nucleic acid molecule sing DNA encoding a light chain as ented by an amino acid sequence of SEQ ID NO: 9, 13, 17 or 21; and (II) a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25, 29 or 33.
In some embodiments, the first nucleic acid le comprises DNA with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the first nucleic acid le selected from (I); the second nucleic acid molecule comprises DNA with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the second nucleic acid molecule selected from (II).
In some embodiments, the nucleotide composition comprises: 1) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9 and a second nucleic acid molecule sing DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25; 2) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid ce of SEQ ID NO: 29; 3) a first nucleic acid molecule comprising DNA ng a light chain as represented by an amino acid sequence of SEQ ID NO: 9 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33; 4) a first nucleic acid molecule sing DNA ng a light chain as represented by an amino acid sequence of SEQ ID NO: 13 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25; ) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid ce of SEQ ID NO: 13 and a second nucleic acid molecule comprising DNA ng a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29; 6) a first nucleic acid molecule comprising DNA encoding a light chain as ented by an amino acid sequence of SEQ ID NO: 13 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33; 7) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25; 8) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 and a second nucleic acid molecule comprising DNA ng a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29; 9) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33; ) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid molecule sing DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25; 11) a first nucleic acid molecule sing DNA ng a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29; 12) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid molecule comprising DNA ng a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33; 13) a first c acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 37 and a second c acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 35; 14) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 39 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 35; ) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 50 and a second nucleic acid le comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 52; or 16) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 54 and a second nucleic acid molecule comprising DNA encoding a heavy chain as ented by an amino acid ce of SEQ ID NO: 56.
In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule comprise DNA with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the first nucleic acid molecule or the second nucleic acid le ed from 1)-16).
In some embodiments, the tide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as ented by an amino acid sequence of SEQ ID NO: 9 and a second c acid molecule sing DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33.
In some ments, the nucleotide composition comprises: a first c acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 13 and a second nucleic acid molecule comprising DNA ng a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule sing DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 13 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 13 and a second c acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 and a second nucleic acid molecule sing DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29.
In some embodiments, the nucleotide composition comprises: a first c acid molecule comprising DNA encoding a light chain as represented by an amino acid ce of SEQ ID NO: 17 and a second nucleic acid le comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA ng a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid le sing DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25.
In some embodiments, the nucleotide ition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33.
In some embodiments, the nucleotide composition comprises: a first nucleic acid le comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 37 and a second nucleic acid molecule sing DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 35.
In some embodiments, the nucleotide composition comprises: a first c acid molecule comprising DNA encoding a light chain as ented by an amino acid sequence of SEQ ID NO: 39 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 35.
In some embodiments, the nucleotide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 50 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 52.
In some embodiments, the tide composition comprises: a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid ce of SEQ ID NO: 54 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 56.
In some embodiments, DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9 is shown as SEQ ID NO: 10.
In some embodiments, DNA encoding a light chain as ented by an amino acid sequence of SEQ ID NO: 13 is shown as SEQ ID NO: 14.
In some embodiments, DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 is shown as SEQ ID NO: 18.
In some embodiments, DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 21 is shown as SEQ ID NO: 22.
In some embodiments, DNA encoding a light chain as represented by an amino acid ce of SEQ ID NO: 37 is shown as SEQ ID NO: 38.
In some embodiments, DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 39 is shown as SEQ ID NO: 40.
In some embodiments, DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 50 is shown as SEQ ID NO: 51.
In some embodiments, DNA ng a light chain as ented by an amino acid sequence of SEQ ID NO: 54 is shown as SEQ ID NO: 55.
In some embodiments, DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25 is shown as SEQ ID NO: 26.
In some embodiments, DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29 is shown as SEQ ID NO: 30.
In some embodiments, DNA ng a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33 is shown as SEQ ID NO: 34.
In some embodiments, DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 35 is shown as SEQ ID NO: 36.
In some embodiments, DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 52 is shown as SEQ ID NO: 53.
In some embodiments, DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 56 is shown as SEQ ID NO: 57.
In yet another respect, the invention provides a vector comprising the nucleotide composition of the invention.
In some embodiments, the vector is a eukaryotic sion vector, a prokaryotic expression vector or a viral vector.
In yet another respect, the invention provides a host cell comprising the vector of the invention.
In some embodiments, the host cell comprising the vector is obtained by vector transformation.
In some embodiments, the host cell is bacteria, yeast or mammalian cell.
In some embodiments, the host cell is escherichia coli, pichia yeast, Chinese r ovary cells or human embryonic kidney 293 cells.
In yet another respect, the invention provides a method of preparing the antibody or n-binding fragment thereof of the ion, comprising expressing the antibody or antigen-binding fragment thereof in the host cell of the invention and isolating the antibody or antigen-binding fragment thereof.
In yet another respect, the invention es a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned isolated antibody or n-binding fragment thereof, and a ceutical acceptable In yet another respect, the invention provides a reagent for detecting LIF in biological samples comprising the above-mentioned isolated antibody or n-binding fragment thereof.
In some embodiments, the biological samples are blood, serum, urine, biopsy materials, tumor, or any s suspected of having abnormal LIF levels.
In another t, the invention provides a method for inhibiting the expression of LIF and/or blocking the activity of LIF, comprising administering to the patient in need thereof a therapeutically effective amount of the above-mentioned isolated antibody or antigen-binding fragment thereof, and/or the above-mentioned pharmaceutical composition.
In yet another respect, the invention provides use of the above-mentioned isolated antibody or antigen-binding fragment thereof, and/or the mentioned pharmaceutical composition in manufacture of a medicament used for inhibiting the expression of LIF and/or ng the activity of LIF.
In yet r respect, the invention provides the above-mentioned isolated antibody or antigen-binding fragment thereof and/or the above-mentioned pharmaceutical composition for use in inhibiting the expression of LIF and/or blocking the activity of LIF.
In another respect, the invention provides a method for treating a disease or condition related to LIF comprising stering to the patient in need a therapeutically effective amount of the above-mentioned isolated dy or antigen-binding fragment, and/or the above-mentioned pharmaceutical composition.
In some embodiments, the e or condition related to LIF is tumor. In some embodiments, the tumor is solid tumor. In some embodiments, the solid tumor comprises astoma, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer or prostate cancer.
In yet another respect, the ion provides use of the above-mentioned isolated antibody or antigen-binding nt thereof, and/or the above-mentioned pharmaceutical composition in manufacture of a medicament for treating a disease or ion related to LIF. In some embodiments, the disease or condition related to LIF is tumor. In some embodiments, the tumor is solid tumor. In some embodiments, the solid tumor ses glioblastoma, lung cancer, n cancer, colorectal cancer, pancreatic cancer or prostate cancer.
In yet another respect, the invention provides the above-mentioned isolated antibody or antigen-binding fragment thereof, and/or the above-mentioned pharmaceutical composition for use in treating a disease or condition related to LIF.
In some embodiments, the disease related to LIF is tumor. In some embodiments, the tumor is solid tumor. In some embodiments, the solid tumor comprises glioblastoma, lung cancer, ovarian cancer, ctal cancer, pancreatic cancer or prostate cancer.
A disease or ion related to LIF means that blocking LIF and LIRR and/or GP130 can treat, alleviate, e and/or stabilize the disease or condition.
In another respect, the invention es a method for detecting the LIF in biological samples comprising (i) obtaining a subject’s tissue or liquid sample, (ii) exposing the tissue or liquid sample to the mentioned isolated antibody or antigen-binding fragment thereof or the above-mentioned reagent; and (iii) comparing the LIF binding to the tissue or liquid sample of (ii) with the LIF binding to a control sample, wherein the increase in the amount of the bound LIF compared with the control sample shows the abnormal level of LIF production, expression or activation.
In some embodiments, the tissue or liquid sample ses blood, serum, urine, biopsy materials, tumor, or any s suspected of having abnormal LIF levels.
Table I Description of the antibody sequence of the invention Sequence No. Sequence Sequence No. Sequence SEQ ID NO:1 (LCDR1 aa of 1C11, humanized RASENIYSYLA anti-LIF antibody or 38E SEQ ID NO:2 (LCDR2 aa of 38E10E1C11, humanized NAKTLAE anti-LIF antibody or 38E Chimeric) SEQ ID NO:3 (LCDR3 aa of 38E10E1C11, humanized QHHYVTPLT IF antibody or 38E Chimeric) SEQ ID NO:4 (HCDR1 aa of 38E10E1C11, humanized SYAMS anti-LIF antibody or 38E Chimeric) SEQ ID NO:5 (HCDR2 aa of humanized anti-LIF TISSGGSNTYSPDTVKG antibody) SEQ ID NO:6 (HCDR3 aa of 38E10E1C11, humanized YYGYYFDF anti-LIF antibody or 38E Chimeric) DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK SEQ ID NO:7 (VL1, aa) PGKSPKLLVYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQHHYVTPLTFGQGTKLEIKR GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCCCGGCAAGAGCCCCAAGCTGCTGGTGT SEQ ID NO:8 (VL1, nt) ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACC TACTACTGCCAGCACCACTACGTGACCCCCCTGACCT TCGGCCAGGGCACCAAGCTGGAGATCAAGAGG DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK LLVYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQ SEQ ID NO:9(full PEDFATYYCQHHYVTPLTFGQGTKLEIKRTVAAPSVFIF light chain 1, aa) PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC Sequence No. Sequence GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCCCGGCAAGAGCCCCAAGCTGCTGGTGT ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACC TACTACTGCCAGCACCACTACGTGACCCCCCTGACCT SEQ ID NO:10(full light TCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCG chain 1, nt) TGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGA CGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTG CCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTG CAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAAC GAGAGCGTGACCGAGCAGGACAGCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC GAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACC AAGAGCTTCAACAGGGGCGAGTGC DIHMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK SEQ ID NO:11 (VL2, aa) PGKSPKLLVYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQ YYCQHHYVTPLTFGQGTKLEIKR GACATCCACATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCCCGGCAAGAGCCCCAAGCTGCTGGTGT SEQ ID NO:12 (VL2, nt) ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACC TACTACTGCCAGCACCACTACGTGACCCCCCTGACCT TCGGCCAGGGCACCAAGCTGGAGATCAAGAGG DIHMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK PGKSPKLLVYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQ SEQ ID NO:13 (full light YYCQHHYVTPLTFGQGTKLEIKRTVAAPSVFIF chain 2, aa) PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC Sequence No. Sequence GACATCCACATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCCCGGCAAGAGCCCCAAGCTGCTGGTGT ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACC TACTACTGCCAGCACCACTACGTGACCCCCCTGACCT SEQ ID NO:14 (full light TCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCG chain 2, nt) CCCCCAGCGTGTTCATCTTCCCCCCCAGCGA CGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTG GAACAACTTCTACCCCAGGGAGGCCAAGGTG CAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAAC AGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGAC TACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC GAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACC AAGAGCTTCAACAGGGGCGAGTGC DIHMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK SEQ ID NO:15 (VL3, aa) PGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFTLTISSLQ PEDFATYYCQHHYVTPLTFGQGTKLEIKR GACATCCACATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCCCGGCAAGAGCCCCCAGCTGCTGGTGT SEQ ID NO:16 (VL3, nt) ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCCAGTTCACCCT GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACC TACTACTGCCAGCACCACTACGTGACCCCCCTGACCT TCGGCCAGGGCACCAAGCTGGAGATCAAGAGG DIHMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK PGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFTLTISSLQ SEQ ID NO:17 (full light PEDFATYYCQHHYVTPLTFGQGTKLEIKRTVAAPSVFIF chain 3, aa) PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC Sequence No. Sequence GACATCCACATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCCCGGCAAGAGCCCCCAGCTGCTGGTGT ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCCAGTTCACCCT GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACC TACTACTGCCAGCACCACTACGTGACCCCCCTGACCT SEQ ID NO:18 (full light TCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCG chain 3, nt) TGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGA CGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTG CCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTG CAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAAC AGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC GAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACC TTCAACAGGGGCGAGTGC DIHMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK SEQ ID NO:19(VL4 QGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFTLKINSL , aa) QPEDFATYYCQHHYVTPLTFGQGTKLEIKR GACATCCACATGACCCAGAGCCCCAGCAGCCTGAGC GTGGGCGACAGGGTGACCATCACCTGCAGG GAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGGGCAAGAGCCCCCAGCTGCTGGTGT SEQ ID NO:20(VL4 ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA , nt) GGTTCAGCGGCAGCGGCAGCGGCACCCAGTTCACCCT GAAGATCAACAGCCTGCAGCCCGAGGACTTCGCCAC CTACTACTGCCAGCACCACTACGTGACCCCCCTGACC TTCGGCCAGGGCACCAAGCTGGAGATCAAGAGG DIHMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK QGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFTLKINSL SEQ ID NO:21(full light QPEDFATYYCQHHYVTPLTFGQGTKLEIKRTVAAPSVFI chain 4 FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL , aa) QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC Sequence No. Sequence GACATCCACATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGT CCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCCAGTTCACCCT GAAGATCAACAGCCTGCAGCCCGAGGACTTCGCCAC CTACTACTGCCAGCACCACTACGTGACCCCCCTGACC SEQ ID NO:22(full light TTCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACC chain 4 GTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCG , nt) ACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGT TGAACAACTTCTACCCCAGGGAGGCCAAGGT GCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAA CAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGA CAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGC GAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACC AAGAGCTTCAACAGGGGCGAGTGC EVMLLESGGGLVQPGGSLRLSCAASGFIFSSYAMS SEQ ID NO:23(VH1 WVRQAPGTGLEWVATISSGGSNTYSPDTVKGRFTIS , aa) RDNSKNTLYLQMNSLRAEDTAVYYCARYYGYYFD FWGQGTLLTVSS GAGGTGATGCTGCTGGAGAGCGGCGGCGGCCTG GTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCG CCGCCAGCGGCTTCATCTTCAGCAGCTACGCCAT GAGCTGGGTGAGGCAGGCCCCCGGCACCGGCCTG GAGTGGGTGGCCACCATCAGCAGCGGCGGCAGC SEQ ID NO:24(VH1 AACACCTACAGCCCCGACACCGTGAAGGGCAGGT , nt) TCACCATCAGCAGGGACAACAGCAAGAACACCCT GTACCTGCAGATGAACAGCCTGAGGGCCGAGGA CACCGCCGTGTACTACTGCGCCAGGTACTACGGC TACTACTTCGACTTCTGGGGCCAGGGCACCCTGC TGACCGTGAGCAGC Sequence No. Sequence EVMLLESGGGLVQPGGSLRLSCAASGFIFSSYAMS WVRQAPGTGLEWVATISSGGSNTYSPDTVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCARYYGYYFD FWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS SEQ ID NO:25(full heavy LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC chain 1 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT , aa) CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY VSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence No. ce GAGGTGATGCTGCTGGAGAGCGGCGGCGGCCTG GTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCG CCGCCAGCGGCTTCATCTTCAGCAGCTACGCCAT GAGCTGGGTGAGGCAGGCCCCCGGCACCGGCCTG GAGTGGGTGGCCACCATCAGCAGCGGCGGCAGC AACACCTACAGCCCCGACACCGTGAAGGGCAGGT TCACCATCAGCAGGGACAACAGCAAGAACACCCT GCAGATGAACAGCCTGAGGGCCGAGGA CACCGCCGTGTACTACTGCGCCAGGTACTACGGC TACTACTTCGACTTCTGGGGCCAGGGCACCCTGC TGACCGTGAGCAGCGCCAGCACCAAGGGCCCCA GCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCAC CAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTG AAGGACTACTTCCCCGAGCCCGTGACCGTGAGCT GGAACAGCGGCGCCCTGACCAGCGGCGTGCACA CCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTA CAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGC GGCACCCAGACCTACATCTGCAACGTGA ACCACAAGCCCAGCAACACCAAGGTGGACAAGA SEQ ID NO:26(full heavy AGCCCAAGAGCTGCGACAAGACCCACA chain 1 CCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGG , nt) CGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCC AAGGACACCCTGATGATCAGCAGGACCCCCGAG GTGACCTGCGTGGTGGTGGACGTGAGCCACGAGG ACCCCGAGGTGAAGTTCAACTGGTACGTGGACGG CGTGGAGGTGCACAACGCCAAGACCAAGCCCAG GGAGGAGCAGTACAACAGCACCTACAGGGTGGT GAGCGTGCTGACCGTGCTGCACCAGGACTGGCTG AACGGCAAGGAGTACAAGTGCAAGGTGAGCAAC AAGGCCCTGCCCGCCCCCATCGAGAAGACCATCA GCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGG TGTACACCCTGCCCCCCAGCAGGGAGGAGATGAC CAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAG GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGG AGAGCAACGGCCAGCCCGAGAACAACTACAAGA CCACCCCCCCCGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACAGCAAGCTGACCGTGGACAAGAGC AGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCG TGATGCACGAGGCCCTGCACAACCACTACACCCA GAAGAGCCTGAGCCTGAGCCCCGGCAAG Sequence No. Sequence EVMLVESGGGLVQPGGSLRLSCAASGFIFSSYAMSWVR SEQ ID NO:27(VH2 QAPGTGLEWVATISSGGSNTYSPDTVKGRFTISRDNSKN , aa) TLYLQMNSLRAEDTAMYYCARYYGYYFDFWGQGTLL GAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTGGTG CAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCC TTCATCTTCAGCAGCTACGCCATGAGCTGGG TGAGGCAGGCCCCCGGCACCGGCCTGGAGTGGGTGG SEQ ID VH2 CCACCATCAGCAGCGGCGGCAGCAACACCTACAGCC ,nt) CCGACACCGTGAAGGGCAGGTTCACCATCAGCAGGG ACAACAGCAAGAACACCCTGTACCTGCAGATGAACA GCCTGAGGGCCGAGGACACCGCCATGTACTACTGCG CCAGGTACTACGGCTACTACTTCGACTTCTGGGGCCA CCTGCTGACCGTGAGCAGC EVMLVESGGGLVQPGGSLRLSCAASGFIFSSYAMSWVR QAPGTGLEWVATISSGGSNTYSPDTVKGRFTISRDNSKN TLYLQMNSLRAEDTAMYYCARYYGYYFDFWGQGTLL TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SEQ ID NO:29(full heavy SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC chain 2 , aa) PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence No. Sequence GAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTGGTG CAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCC AGCGGCTTCATCTTCAGCAGCTACGCCATGAGCTGGG TGAGGCAGGCCCCCGGCACCGGCCTGGAGTGGGTGG CCACCATCAGCAGCGGCGGCAGCAACACCTACAGCC CCGACACCGTGAAGGGCAGGTTCACCATCAGCAGGG ACAACAGCAAGAACACCCTGTACCTGCAGATGAACA GCCTGAGGGCCGAGGACACCGCCATGTACTACTGCG CCAGGTACTACGGCTACTACTTCGACTTCTGGGGCCA GGGCACCCTGCTGACCGTGAGCAGCGCCAGCACCAA GGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAG AGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTG GTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCT GGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCT TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCT GAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGG CACCCAGACCTACATCTGCAACGTGAACCACAAGCCC SEQ ID NO:30 (full heavy AGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG chain 2 AGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCG , nt) CCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTT CAAGCCCAAGGACACCCTGATGATCAGCAG CGAGGTGACCTGCGTGGTGGTGGACGTGAG CCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCC CAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGT GAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAA CGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGC CCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGC CCAGCCCAGGGAGCCCCAGGTGTACACCCT GCCCCCCAGCAGGGAGGAGATGACCAAGAACCAGGT GACCTGCCTGGTGAAGGGCTTCTACCCCAGC GACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCC GAGAACAACTACAAGACCACCCCCCCCGTGCTGGAC AGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCG TGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACT ACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG EVMLVESGGGLVQPGGSLRLSCAASGFIFSSYAMS SEQ ID NO:31 (VH3 WVRQAPETRLEWVATISSGGSNTYSPDTVKGRFTIS , aa) RDNSKNTLYLQMNSLRAEDTAMYYCARYYGYYFD FWGQGTLLTVSS Sequence No. Sequence GAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTG GTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCG CCGCCAGCGGCTTCATCTTCAGCAGCTACGCCAT GAGCTGGGTGAGGCAGGCCCCCGAGACCAGGCT GGAGTGGGTGGCCACCATCAGCAGCGGCGGCAG SEQ ID VH3 CAACACCTACAGCCCCGACACCGTGAAGGGCAG ,nt) GTTCACCATCAGCAGGGACAACAGCAAGAACAC CCTGCAGATGAACAGCCTGAGGGCCGAG GACACCGCCATGTACTACTGCGCCAGGTACTACG GCTACTACTTCGACTTCTGGGGCCAGGGCACCCT GCTGACCGTGAGCAGC EVMLVESGGGLVQPGGSLRLSCAASGFIFSSYAMS WVRQAPETRLEWVATISSGGSNTYSPDTVKGRFTIS RDNSKNTLYLQMNSLRAEDTAMYYCARYYGYYFD FWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS SEQ ID NO:33(full heavy LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC chain 3 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT , aa) CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence No. ce ATGCTGGTGGAGAGCGGCGGCGGCCTGGTG CAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCC AGCGGCTTCATCTTCAGCAGCTACGCCATGAGCTGGG TGAGGCAGGCCCCCGAGACCAGGCTGGAGTGGGTGG CCACCATCAGCAGCGGCGGCAGCAACACCTACAGCC CCGACACCGTGAAGGGCAGGTTCACCATCAGCAGGG ACAACAGCAAGAACACCCTGTACCTGCAGATGAACA GCCTGAGGGCCGAGGACACCGCCATGTACTACTGCG CCAGGTACTACGGCTACTACTTCGACTTCTGGGGCCA CCTGCTGACCGTGAGCAGCGCCAGCACCAA GGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAG AGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTG GTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCT GGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCT TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCT GAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGG CACCCAGACCTACATCTGCAACGTGAACCACAAGCCC SEQ ID NO:34(full heavy AGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG chain 3 AGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCG , nt) CCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTT CCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAG GACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAG CCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCC CAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGT GAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAA CGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGC CCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGC CAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCT CAGCAGGGAGGAGATGACCAAGAACCAGGT GAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGC GACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCC GAGAACAACTACAAGACCACCCCCCCCGTGCTGGAC AGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCG TGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACT ACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG Sequence No. Sequence EVMLVESGGGLVQPGGSLRLSCAASGFIFSSYAMSWVR QAPETRLEWVATISSGGSNTYSPDTVKGRFTISRDNSKN TLYLQMNSLRAEDTAMYYCARYYGYYFDFWGQGTLL TVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFP SEQ ID NO:35(38E WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSS HuH3L2-m or 38E TWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVP HuH3L4-m full heavy EVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQF chain,aa) SWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDW LNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPP PKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAEN YKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSV LHEGLHNHHTEKSLSHSPGK Sequence No. Sequence GAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTGGTG CAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCC AGCGGCTTCATCTTCAGCAGCTACGCCATGAGCTGGG TGAGGCAGGCCCCCGAGACCAGGCTGGAGTGGGTGG CCACCATCAGCAGCGGCGGCAGCAACACCTACAGCC CCGACACCGTGAAGGGCAGGTTCACCATCAGCAGGG ACAACAGCAAGAACACCCTGTACCTGCAGATGAACA GGGCCGAGGACACCGCCATGTACTACTGCG CCAGGTACTACGGCTACTACTTCGACTTCTGGGGCCA GGGCACCCTGCTGACCGTGAGCAGCGCCAAGACCAC CCCCCCCAGCGTGTACCCCCTGGCCCCCGGCAGCGCC GCCCAGACCAACAGCATGGTGACCCTGGGCTGCCTG GTGAAGGGCTACTTCCCCGAGCCCGTGACCGTGACCT GGAACAGCGGCAGCCTGAGCAGCGGCGTGCACACCT TCCCCGCCGTGCTGCAGAGCGACCTGTACACCCTGAG CAGCAGCGTGACCGTGCCCAGCAGCACCTGGCCCAG CGTGACCTGCAACGTGGCCCACCCCGCCAG SEQ ID NO:36(38E CAGCACCAAGGTGGACAAGAAGATCGTGCCCAGGGA HuH3L2-m or 38E CTGCGGCTGCAAGCCCTGCATCTGCACCGTGCCCGAG HuH3L4-m full heavy chain, GTGAGCAGCGTGTTCATCTTCCCCCCCAAGCCCAAGG ACGTGCTGACCATCACCCTGACCCCCAAGGTGACCTG CGTGGTGGTGGACATCAGCAAGGACGACCCCGAGGT GCAGTTCAGCTGGTTCGTGGACGACGTGGAGGTGCAC ACCGCCCAGACCCAGCCCAGGGAGGAGCAGTTCAAC AGCACCTTCAGGAGCGTGAGCGAGCTGCCCATCATGC ACCAGGACTGGCTGAACGGCAAGGAGTTCAAGTGCA GGGTGAACAGCGCCGCCTTCCCCGCCCCCATCGAGAA CAGCAAGACCAAGGGCAGGCCCAAGGCCCC CCAGGTGTACACCATCCCCCCCCCCAAGGAGCAGATG GCCAAGGACAAGGTGAGCCTGACCTGCATGATCACC GACTTCTTCCCCGAGGACATCACCGTGGAGTGGCAGT GGAACGGCCAGCCCGCCGAGAACTACAAGAACACCC AGCCCATCATGGACACCGACGGCAGCTACTTCGTGTA CAGCAAGCTGAACGTGCAGAAGAGCAACTGGGAGGC CGGCAACACCTTCACCTGCAGCGTGCTGCACGAGGGC CTGCACAACCACCACACCGAGAAGAGCCTGAGCCAC AGCCCCGGCAAG DIHMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK LLVYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQ SEQ ID NO:37 (38E PEDFATYYCQHHYVTPLTFGQGTKLEIKRADAAPTVSIF HuH3L2-m full light chain, PPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQN GVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC Sequence No. Sequence GACATCCACATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCCCGGCAAGAGCCCCAAGCTGCTGGTGT ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCT GACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACC TACTACTGCCAGCACCACTACGTGACCCCCCTGACCT SEQ ID NO:38 (38E TCGGCCAGGGCACCAAGCTGGAGATCAAGAGGGCCG HuH3L2-m full light chain, ACGCCGCCCCCACCGTGAGCATCTTCCCCCCCAGCAG CGAGCAGCTGACCAGCGGCGGCGCCAGCGTGGTGTG GAACAACTTCTACCCCAAGGACATCAACGTG AAGTGGAAGATCGACGGCAGCGAGAGGCAGAACGGC GTGCTGAACAGCTGGACCGACCAGGACAGCAAGGAC AGCACCTACAGCATGAGCAGCACCCTGACCCTGACC GAGTACGAGAGGCACAACAGCTACACCTGC GAGGCCACCCACAAGACCAGCACCAGCCCCATCGTG AAGAGCTTCAACAGGAACGAGTGC DIHMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQK QGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFTLKINSL SEQ ID NO:39(38E QPEDFATYYCQHHYVTPLTFGQGTKLEIKRADAAPTVSI HuH3L4-m full light chain, FPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC EATHKTSTSPIVKSFNRNEC GACATCCACATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGT ACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCCAGTTCACCCT GAAGATCAACAGCCTGCAGCCCGAGGACTTCGCCAC CTACTACTGCCAGCACCACTACGTGACCCCCCTGACC SEQ ID NO:40 (38E TTCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACC HuH3L4-m full light chain, GTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCG ACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGT GCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGT GCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAA CAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGA CAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC GACTACGAGAAGCACAAGGTGTACGCCTGC GAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACC AAGAGCTTCAACAGGGGCGAGTGC Sequence No. ce DIHMTQSPASLSASVGETVTITCRASENIYSYLAWYQQK LLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSL SEQ ID NO:41 QPEDFGSYYCQHHYVTPLTFGAGTKLELKRADAAPTVS (38E10E1C11 full light IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER chain aa) QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYT CEATHKTSTSPIVKSFNRNEC GACATCCACATGACTCAGTCTCCAGCCTCCCTATCTG CATCTGTGGGAGAAACTGTCACCATCACATGTCGAGC AAGTGAGAATATTTACAGTTATTTAGCATGGTATCAG CAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATA ATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGT TCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAA GATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTAT CAACATCATTATGTTACTCCGCTCACGTTCGG SEQ ID NO:42 TGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGC (38E10E1C11 full light TGCACCAACTGTATCCATCTTCCCACCATCCAGTGAG chain nt) CAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCT ACTTCTACCCCAAAGACATCAATGTCAAGTG GAAGATTGATGGCAGTGAACGACAAAATGGCGTCCT GAACAGTTGGACTGATCAGGACAGCAAAGACAGCAC CTACAGCATGAGCAGCACCCTCACGTTGACCAAGGA CGAGTATGAACGACATAACAGCTATACCTGTGAGGC CACTCACAAGACATCAACTTCACCCATTGTCAAGAGC TTCAACAGGAATGAGTGT EVMLVESGGGLVKPGGSLKLSCAASGFIFSSYAMSWVR QSPETRLEWVATISSGGSNTYSPDSVKGRFTISRDNAKN TLYLQMSSLRSEDTAMYYCARYYGYYFDFWGQGTTLT VSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTW SEQ ID NO:43 PSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEV (38E10E1C11 full heavy SSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSW chain aa) FVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLN GKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYK NTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHE GLHNHHTEKSLSHSPGK Sequence No. Sequence GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTG AAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCT CTGGATTCATTTTCAGTAGTTATGCCATGTCTTGGGTT CGCCAGAGTCCGGAGACGAGGCTGGAGTGGGTCGCA ACCATTAGTAGTGGTGGTAGTAACACCTACTCTCCAG TGAAGGGGCGATTCACCATCTCCAGAGACA ATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCT GAGGTCTGAGGACACGGCCATGTATTACTGTGCAAG ATATTATGGTTACTACTTTGACTTCTGGGGCCAAGGC ACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCC CATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCA AACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAG GGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACT CTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGC TGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCA GTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCG TCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAA SEQ ID CAAGAAAATTGTGCCCAGGGATTGTGGTTGT NO:44(38E10E1C11 full AAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTG heavy chain nt) TCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCAC CATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTA GACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCT GGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGAC GCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCG CAGTGAACTTCCCATCATGCACCAGGACTGG CTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGT GCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCA AAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACA CACCTCCCAAGGAGCAGATGGCCAAGGATA AAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCC TGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCA GCCAGCGGAGAACTACAAGAACACTCAGCCCATCAT GGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTC AATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACT TTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACC ACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAA SEQ ID NO:45 (38E10E1C11 或 38E TISSGGSNTYSPDSVKG Chimeric HCDR2 aa) DIHMTQSPASLSASVGETVTITCRASENIYSYLAWYQQK SEQ ID NO:46 (38E QGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSL Chimeric VL aa) QPEDFGSYYCQHHYVTPLTFGAGTKLELKR Sequence No. Sequence GACATCCACATGACTCAGTCTCCAGCCTCCCTATCTG CATCTGTGGGAGAAACTGTCACCATCACATGTCGAGC GAATATTTACAGTTATTTAGCATGGTATCAG CAGAAACAGGGAAAATCTCCTCAGCTCCTGTCTATAA SEQ ID NO:47 (38E TGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTC Chimeric VL nt) AGTGGCAGTGGATCAGGCACACATTTTCTCTGAAGAT CAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTAC CATCATTATGTTACTCCGCTCACGTTCGGTGC TGGGACCAAGCTGGAGCTGAAACGGGC EVMLVESGGGLVKPGGSLKLSCAASGFIFSSYAMSWVR SEQ ID NO:48 (38E QSPETRLEWVATISSGGSNTYSPDSVKGRFTISRDNAKN Chimeric VH aa) TLYLQMSSLRSEDTAMYYCARYYGYYFDFWGQGTTLT GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTG AAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCT CTGGATTCATTTTCAGTAGTTATGCCATGTCTTGGGTT CGCCAGAGTCCGGAGACGAGGCTGGAGTGGGTCGCA SEQ ID NO:49 (38E ACCATTAGTAGTGGTGGTAGTAACACCTACTCTCCAG Chimeric VH nt) ACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACA ATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCT GAGGTCTGAGGACACGGCCATGTATTACTGTGCAAG ATATTATGGTTACTACTTTGACTTCTGGGGCCAAGGC ACCACTCTCACAGTCTCCTCA DIHMTQSPASLSASVGETVTITCRASENIYSYLAWYQQK QGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSL SEQ ID NO:50 QPEDFGSYYCQHHYVTPLTFGAGTKLELKRTVAAPSVFI (38E ic full light FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL chain aa) QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC Sequence No. Sequence GACATCCACATGACCCAGAGCCCCGCCAGCCTGAGC GCCAGCGTGGGCGAGACCGTGACCATCACCTGCAGG GCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACC AGCAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGT CCAAGACCCTGGCCGAGGGCGTGCCCAGCA GGTTCAGCGGCAGCGGCAGCGGCACCCAGTTCAGCC TGAAGATCAACAGCCTGCAGCCCGAGGACTTCGGCA GCTACTACTGCCAGCACCACTACGTGACCCCCCTGAC SEQ ID NO:51 CTTCGGCGCCGGCACCAAGCTGGAGCTGAAGAGGAC (38E Chimeric full light CGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGC chain nt) GACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTG TGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGG TGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCA ACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGG ACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGA GCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCT GCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGA CCAAGAGCTTCAACAGGGGCGAGTGC SGGGLVKPGGSLKLSCAASGFIFSSYAMSWVR QSPETRLEWVATISSGGSNTYSPDSVKGRFTISRDNAKN TLYLQMSSLRSEDTAMYYCARYYGYYFDFWGQGTTLT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS SEQ ID NO:52 LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP (38E Chimeric full heavy APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE chain aa) DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK Sequence No. Sequence GAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTGGTG GGCGGCAGCCTGAAGCTGAGCTGCGCCGCC AGCGGCTTCATCTTCAGCAGCTACGCCATGAGCTGGG TGAGGCAGAGCCCCGAGACCAGGCTGGAGTGGGTGG CCACCATCAGCAGCGGCGGCAGCAACACCTACAGCC CCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGG CCAAGAACACCCTGTACCTGCAGATGAGCA GCCTGAGGAGCGAGGACACCGCCATGTACTACTGCG CCAGGTACTACGGCTACTACTTCGACTTCTGGGGCCA GGGCACCACCCTGACCGTGAGCAGCGCCAGCACCAA GGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAG AGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTG GTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCT GGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCT TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCT GAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGG GACCTACATCTGCAACGTGAACCACAAGCCC SEQ ID NO:53 AGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG (38E Chimeric full heavy AGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCG chain nt) CCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTT CCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAG GACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAG CCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCC CAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGT GAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAA GGAGTACAAGTGCAAGGTGAGCAACAAGGC CCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGC CAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCT GCCCCCCAGCAGGGAGGAGATGACCAAGAACCAGGT GAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGC GACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCC GAGAACAACTACAAGACCACCCCCCCCGTGCTGGAC AGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCG TGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACT ACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG DIVMTQSHKFMSTSVGDRVSITCKASQDVSNAVAWYQ QKPGQSPRLLIYWASFRHTGVPDRFTGSGSGTEYTLTIS SEQ ID NO:54 RVQAEDLALYYCQQHYNTPYTFGGGTRLEIKRADAAPT (P36-033 full light chain aa) VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSE RQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNS YTCEATHKTSTSPIVKSFNRNEC Sequence No. Sequence GACATCGTGATGACCCAGTCCCACAAGTTCATGAGCA CCAGCGTGGGCGATCGGGTGTCCATCACCTGTAAGGC CTCCCAGGACGTGAGCAACGCCGTGGCCTGGTATCAG CAGAAGCCTGGCCAGTCCCCTCGGCTGCTGATCTATT GGGCTTCCTTCAGGCACACCGGCGTGCCCGATCGGTT CACCGGCTCCGGATCCGGCACCGAGTATACCCTGACC CGGGTGCAGGCCGAGGATCTGGCTCTGTATT ATTGTCAGCAGCACTACAATACCCCCTACACCTTCGG SEQ ID NO:55 CGGCGGCACCAGGCTGGAGATCAAGAGAGCTGATGC (P36-033 full light chain nt) TGCCCCCACCGTGAGCATCTTCCCTCCCTCCAGCGAG ACCTCCGGCGGAGCCTCCGTGGTGTGCTTCC TGAACAACTTCTACCCCAAGGATATCAACGTGAAGTG GAAGATCGACGGCAGCGAGCGGCAGAATGGCGTGCT GAACTCCTGGACCGACCAGGACAGCAAGGACTCCAC CTATTCCATGTCCTCCACCCTGACCCTGACCAAGGAT GAGTACGAGCGGCACAACAGCTATACCTGTGAGGCC ACCCACAAGACCTCCACCTCCCCCATCGTGAAGTCCT TCAATAGGAATGAGTGC EVMLVESGGGLVQPGGSRRLSCAASGFTFSSYPMFWVR MEWVAYISNGGDSTYYPDTVKGRFTVSRDNA KNTLYLQMSSLKSVDTAIYYCVRPSARYDEWFAYWGQ GTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVT SEQ ID NO:56 VPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI (P36-033 full heavy chain CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDP EVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMH QDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQV YTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQ PAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTF TCSVLHEGLHNHHTEKSLSHSPGK Sequence No. Sequence GAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTGGTG CAGCCCGGCGGCAGCAGGAGGCTGAGCTGCGCCGCC AGCGGCTTCACCTTCAGCAGCTACCCCATGTTCTGGG TGAGGCAGACCCCCGAGAAGAGGATGGAGTGGGTGG CCTACATCAGCAACGGCGGCGACAGCACCTACTACCC CGACACCGTGAAGGGCAGGTTCACCGTGAGCAGGGA CAACGCCAAGAACACCCTGTACCTGCAGATGAGCAG CCTGAAGAGCGTGGACACCGCCATCTACTACTGCGTG AGGCCCAGCGCCAGGTACGACGAGTGGTTCGCCTACT AGGGCACCCTGGTGACCGTGAGCAGCGCCA AGACCACCCCCCCCAGCGTGTACCCCCTGGCCCCCGG CAGCGCCGCCCAGACCAACAGCATGGTGACCCTGGG CTGCCTGGTGAAGGGCTACTTCCCCGAGCCCGTGACC TGGAACAGCGGCAGCCTGAGCAGCGGCGTG CACACCTTCCCCGCCGTGCTGCAGAGCGACCTGTACA CCCTGAGCAGCAGCGTGACCGTGCCCAGCAGCACCT GCGAGACCGTGACCTGCAACGTGGCCCACC SEQ ID NO:57 CCGCCAGCAGCACCAAGGTGGACAAGAAGATCGTGC (P36-033 full heavy chain CCAGGGACTGCGGCTGCAAGCCCTGCATCTGCACCGT nt) GCCCGAGGTGAGCAGCGTGTTCATCTTCCCCCCCAAG CCCAAGGACGTGCTGACCATCACCCTGACCCCCAAGG GCGTGGTGGTGGACATCAGCAAGGACGACC CCGAGGTGCAGTTCAGCTGGTTCGTGGACGACGTGGA GGTGCACACCGCCCAGACCCAGCCCAGGGAGGAGCA GTTCAACAGCACCTTCAGGAGCGTGAGCGAGCTGCCC ATCATGCACCAGGACTGGCTGAACGGCAAGGAGTTC AAGTGCAGGGTGAACAGCGCCGCCTTCCCCGCCCCCA TCGAGAAGACCATCAGCAAGACCAAGGGCAGGCCCA AGGCCCCCCAGGTGTACACCATCCCCCCCCCCAAGGA GCAGATGGCCAAGGACAAGGTGAGCCTGACCTGCAT GATCACCGACTTCTTCCCCGAGGACATCACCGTGGAG TGGCAGTGGAACGGCCAGCCCGCCGAGAACTACAAG AACACCCAGCCCATCATGGACACCGACGGCAGCTAC TTCGTGTACAGCAAGCTGAACGTGCAGAAGAGCAAC TGGGAGGCCGGCAACACCTTCACCTGCAGCGTGCTGC ACGAGGGCCTGCACAACCACCACACCGAGAAGAGCC TGAGCCACAGCCCCGGCAAG MKVLAAGVVPLLLVLHWKHGAGSPLPITPVNATCAIRH PCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPN SEQ ID NO:58 NLDKLCGPNVTDFPPFHANGTEKAKLVELYRIVVYLGT (Human LIF aa) SLGNITRDQKILNPSALSLHSKLNATADILRGLLSNVLCR LCSKYHVGHVDVTYGPDTSGKDVFQKKKLGCQLLGKY KQIIAVLAQAF ce No. Sequence MKVLAAGVVPLLLVLHWKHGAGSPLPITPVNATCAIRH PCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPN SEQ ID NO:59 NLDKLCGPNVTDFPPFHANGTEKAKLVELYRIVVYLGT (Mut3 aa) SLGNITRDQKILNPSALSLHSKLNATADILRGLLSNVLCR LCSKYHVGHVDVTYGPDTSGKDVFQKKKLGCQLLGTY KQVISVVVQAF MKVLAAGVVPLLLVLHWKHGAGSPLPITPVNATCAIRH PCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPN SEQ ID NO:60 NLDKLCGPNVTDFPPFHANGTEKAKLVELYRIVVYLGT (Mut4 aa) RDQKILNPSALSLHSKLNATADILRGLLSNVLCR LCSKYHVGHVDVPPVPDHSDKEAFQRKKLGCQLLGTY KQVISVVVQAF SEQ ID NO:61 TYGPDTSGKDVFQKK QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWV RQAPGKGLEWVGQIKAKSDDYATYYAESVKGRFTISRD DSKNTLYLQMNSLKTEDTAVYYCTCWEWDLDFWGQG TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV SEQ ID NO:62 PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP (5D8 full heavy chain aa) PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence No. Sequence CAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGTG AAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCC AGCGGCTTCACCTTCAGCCACGCCTGGATGCACTGGG AGGCCCCCGGCAAGGGCCTGGAGTGGGTGG GCCAGATCAAGGCCAAGAGCGACGACTACGCCACCT ACTACGCCGAGAGCGTGAAGGGCAGGTTCACCATCA GCAGGGACGACAGCAAGAACACCCTGTACCTGCAGA TGAACAGCCTGAAGACCGAGGACACCGCCGTGTACT CCTGCTGGGAGTGGGACCTGGACTTCTGGGG CCAGGGCACCATGGTGACCGTGAGCAGCGCCAGCAC CAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGC AAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGA GCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACA CCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAG CCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCT GGGCACCCAGACCTACATCTGCAACGTGAACCACAA GCCCAGCAACACCAAGGTGGACAAGAGGGTGGAGCC SEQ ID NO:63 CAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGC (5D8 full heavy chain nt) CCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCC TGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAG CCCCGAGGTGACCTGCGTGGTGGTGGACGT GAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAA GCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGT GGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTG AACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAG GCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAG GCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACC CTGCCCCCCAGCAGGGAGGAGATGACCAAGAACCAG GTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGC CCGAGAACAACTACAAGACCACCCCCCCCGTGCTGG ACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGAC CGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTT CAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCA CTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAA Sequence No. ce DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGHTYLN WLQQRPGQPPRLLIYSVSNLESGVPDRFSGSGAGTDFTL SEQ ID NO:64 KISRVEAEDVGVYYCMQATHAPPYTFGQGTKLEIKRTV (5D8 full light chain aa) AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC GACATCGTGATGACCCAGACCCCCCTGAGCAGCCCCG TGACCCTGGGCCAGCCCGCCAGCATCAGCTGCAGGA GCAGCCAGAGCCTGCTGGACAGCGACGGCCACACCT ACCTGAACTGGCTGCAGCAGAGGCCCGGCCAGCCCC CCAGGCTGCTGATCTACAGCGTGAGCAACCTGGAGA GCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCGCCG GCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGG CCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCA CCCACGCCCCCCCCTACACCTTCGGCCAGGGCACCAA SEQ ID NO:65 GCTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGT (5D8 full light chain nt) CTTCCCCCCCAGCGACGAGCAGCTGAAGAGC GGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCT ACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACA ACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGA CCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGA GCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGA AGGTGTACGCCTGCGAGGTGACCCACCAGG GCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGG GCGAGTGC SEQ ID NO:66 KASQDVSNAVA (P36-033 LCDR1 aa) SEQ ID NO:67 WASFRHT (P36-033 LCDR2 aa) SEQ ID NO:68 QQHYNTPYT (P36-033 LCDR3 aa) SEQ ID NO:69 SYPMF (P36-033 HCDR1 aa) SEQ ID NO:70 YISNGGDSTYYPDTVKG (P36-033 HCDR2 aa) SEQ ID NO:71 PSARYDEWFAY (P36-033 HCDR3 aa) Sequence No. Sequence GACATCGTGATGACCCAGTCCCACAAGTTCATGAGCA CCAGCGTGGGCGATCGGGTGTCCATCACCTGTAAGGC CTCCCAGGACGTGAGCAACGCCGTGGCCTGGTATCAG CAGAAGCCTGGCCAGTCCCCTCGGCTGCTGATCTATT SEQ ID NO:72 CCTTCAGGCACACCGGCGTGCCCGATCGGTT 33 VL nt) CACCGGCTCCGGATCCGGCACCGAGTATACCCTGACC ATCTCCCGGGTGCAGGCCGAGGATCTGGCTCTGTATT ATTGTCAGCAGCACTACAATACCCCCTACACCTTCGG CGGCGGCACCAGGCTGGAGATCAAG GAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTGGTG CAGCCTGGAGGATCTCGGAGGCTGAGCTGTGCCGCC AGCGGCTTCACCTTCTCCTCCTATCCCATGTTCTGGGT GACCCCCGAGAAGCGGATGGAGTGGGTGGC SEQ ID NO:73 CTATATCTCCAATGGCGGCGATTCCACCTATTATCCT (P36-033 VH nt) GACACCGTGAAGGGCCGGTTCACCGTGAGCCGGGAT AACGCCAAGAATACCCTGTACCTGCAGATGAGCAGC CTGAAGTCCGTGGACACCGCTATCTACTATTGCGTGA GGCCCTCCGCTCGGTACGACGAGTGGTTCGCCTATTG GGGCCAGGGCACCCTGGTGACAGTGAGCGCT DIHMTQSPASLSASVGETVTITCRASENIYSYLAWYQQK SEQ ID NO:74 QGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSL (38E10E1C11 VL aa) QPEDFGSYYCQHHYVTPLTFGAGTKLELKRA EVMLVESGGGLVKPGGSLKLSCAASGFIFSSYAMSWVR SEQ ID NO:75 QSPETRLEWVATISSGGSNTYSPDSVKGRFTISRDNAKN (38E10E1C11 VH aa) TLYLQMSSLRSEDTAMYYCARYYGYYFDFWGQGTTLT CACATGACTCAGTCTCCAGCCTCCCTATCTG CATCTGTGGGAGAAACTGTCACCATCACATGTCGAGC AAGTGAGAATATTTACAGTTATTTAGCATGGTATCAG CAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATA SEQ ID NO:76 ATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGT (38E10E1C11 VL nt) TCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAA GATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTAT TACTGTCAACATCATTATGTTACTCCGCTCACGTTCGG TGCTGGGACCAAGCTGGAGCTGAAACGGGCT Sequence No. Sequence GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTG AAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCT CTGGATTCATTTTCAGTAGTTATGCCATGTCTTGGGTT CGCCAGAGTCCGGAGACGAGGCTGGAGTGGGTCGCA SEQ ID NO:77 ACCATTAGTAGTGGTGGTAGTAACACCTACTCTCCAG (38E10E1C11 VH nt) ACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACA AGAACACCCTGTACCTGCAAATGAGCAGTCT GAGGTCTGAGGACACGGCCATGTATTACTGTGCAAG ATATTATGGTTACTACTTTGACTTCTGGGGCCAAGGC ACCACTCTCACAGTCTCCTCA QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWV SEQ ID NO:78 RQAPGKGLEWVGQIKAKSDDYATYYAESVKGRFTISRD (5D8 VH aa) DSKNTLYLQMNSLKTEDTAVYYCTCWEWDLDFWGQG TMVTVSS DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGHTYLN SEQ ID NO:79 WLQQRPGQPPRLLIYSVSNLESGVPDRFSGSGAGTDFTL (5D8 VL aa) KISRVEAEDVGVYYCMQATHAPPYTFGQGTKLEIKRTV CAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGTG AAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCC AGCGGCTTCACCTTCAGCCACGCCTGGATGCACTGGG TGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGG SEQ ID NO:80 GCCAGATCAAGGCCAAGAGCGACGACTACGCCACCT (5D8 VH nt) ACTACGCCGAGAGCGTGAAGGGCAGGTTCACCATCA GCAGGGACGACAGCAAGAACACCCTGTACCTGCAGA TGAACAGCCTGAAGACCGAGGACACCGCCGTGTACT ACTGCACCTGCTGGGAGTGGGACCTGGACTTCTGGGG CCAGGGCACCATGGTGACCGTGAGCAGC GACATCGTGATGACCCAGACCCCCCTGAGCAGCCCCG TGACCCTGGGCCAGCCCGCCAGCATCAGCTGCAGGA GCAGCCAGAGCCTGCTGGACAGCGACGGCCACACCT ACCTGAACTGGCTGCAGCAGAGGCCCGGCCAGCCCC SEQ ID NO:81 CCAGGCTGCTGATCTACAGCGTGAGCAACCTGGAGA (5D8 VL nt) GCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCGCCG GCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGG CCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCA CCCCCCCCTACACCTTCGGCCAGGGCACCAA GCTGGAGATCAAGAGGACCGTG SEQ ID NO:82 DIVMTQSHKFMSTSVGDRVSITCKASQDVSNAVAWYQ 33 VL aa) QKPGQSPRLLIYWASFRHTGVPDRFTGSGSGTEYTLTIS RVQAEDLALYYCQQHYNTPYTFGGGTRLEIK Sequence No. Sequence SEQ ID NO:83 EVMLVESGGGLVQPGGSRRLSCAASGFTFSSYPMFWVR (P36-033 VH aa) MEWVAYISNGGDSTYYPDTVKGRFTVSRDNA KNTLYLQMSSLKSVDTAIYYCVRPSARYDEWFAYWGQ GTLVTVSA Notes: All amino acid s of CDRs and framework regions are annotated according to the EU index of the Kabat system (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). All sequences do not include Signal peptide. aa: amino acid, nt: nucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS depicts the g ability of the LIF dy of the invention to human LIF protein; depicts the binding y of the LIF antibody of the invention to mouse LIF depicts the binding ability of the LIF antibody of the invention to machin LIF protein; depicts the affinity of the 1C11 antibody of the invention binding to the n; depicts the ability of the 38E10E1C11 antibody of the invention competing with LIFR for binding to human LIF protein; depicts the ability of the P36-033 antibody competing with GP130 receptor for binding to human LIF protein; s the cross-reaction of the 38E10E1C11 antibody and the P36-033 dy of the invention with the IL-6 family protein; depicts that the 38E10E1C11R antibody of the invention recognizes denatured human LIF protein; Subfigure A in depicts that the 38E10E1C11 antibody of the invention inhibits the phosphorylation of colon cancer cells (HCT116) induced by human LIF protein; Subfigure B in depicts that the 38E10E1C11 antibody of the invention inhibits the phosphorylation of STAT3 in pancreatic cancer cells (KP4) d by human LIF protein; s that the 38E10E1C11 antibody of the invention blocks the activation of STAT3 in pancreatic cancer cells KP4 by human LIF secreted by CT26-hLIF cells; depicts that the P36-033 antibody of the invention inhibits the phosphorylation of STAT3 in colon cancer cells (HCT116) induced by human LIF protein; depicts that the 38E1E1C11 and P36-033 antibodies of the invention reverse the proliferation inhibition of M1 cells caused by LIF; depicts that the 38E1E1C11 antibody of the invention inhibits the growth of CT26-hLIF cells in BABL/c mice; depicts the sensitivity of three human pancreatic cancer cell lines to stimulation by human LIF protein; depicts that the 38E10E1C11R dy of the invention inhibits the phosphorylation of STAT3 in KP4 cells stimulated by LIF protein; Subfigure A in depicts the experimental result of the recognition of full-length human LIF n as well as heterozygous LIF protein by the 38E10E1C11 dy of the invention, and Subfigure B in depicts the mental result that 38E10E1C11 antibody of the invention can reverse M1 cell proliferation inhibition caused by full-length human LIF protein and heterozygous LIF protein; A - B depict the experimental result of the antigen binding properties of the humanized anti-LIF antibody of the invention; depicts the mental result of the non-specific binding affinity of the humanized anti-LIF antibody of the invention; depicts the experimental result of the humanized anti-LIF antibody of the invention competing with LIFR for binding human LIF protein; depicts the experimental result of the zed anti-LIF antibody of the invention competing with GP130 to bind human LIF protein; depicts the experimental result of the humanized anti-LIF antibody of the invention recognizing antigen specificity; Subfigure A in depicts the experimental result of the humanized IF antibody of the present invention izing full-length human LIF proteins and heterozygous LIF proteins; and Subfigure B in depicts the experimental result of the humanized anti-LIF antibody of the invention blocking the inhibition of M1 cells proliferation by ength LIF protein and hybrid protein; depicts the mental result of humanized IF antibody of the invention inhibiting the phosphorylation of STAT3 induced by LIF protein; depicts the experimental result that the humanized anti-LIF antibody of the invention can e the inhibition of M1 cell proliferation by human LIF protein. depicts LIF protein-induced phosphorylation level of STAT3 inhibited by humanized anti-LIF antibody and total STAT3 level, detected by HTFR method. depicts ADCC effect of humanized anti-LIF dy.
DETAILED DESCRIPTION Definitions In order that the present description may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the ed description.
The term "LIF" 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 LIF, mouse LIF (Ref Seq 039537.2), or machin LIF (XM_015457518.1). As described elsewhere herein, LIF can be recombinant and/or glycosylated or non-glycosylated.
The term "antibody" as used herein may include whole antibodies and any n binding fragments (i.e., "antigen-binding portions") or single chains thereof.
An "antibody" refers, in one embodiment, to a glycoprotein or an antigen binding portion thereof comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some naturally occurring IgG, IgD and IgA dies, the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. In some naturally occurring antibodies, each light chain is comprised of a light chain le region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL s can be r subdivided into s of hypervariability, termed complementarity determining regions (CDR), and regions that are more conserved, termed framework s (FR), both of which are intermingled arrangement. Herein, the CDRs of the VH region are abbreviated as HCDR, that is, the three CDRs of the VH region can be abbreviated as HCDR1, HCDR2, and HCDR3; the CDRs of the VL region are abbreviated as LCDR, that is, the three CDRs of the VL region can be abbreviated as LCDR1, LCDR2.
LCDR3.Each VH and VL is composed of three CDRs and four FRs, ed from amino-terminus to y-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the dies may mediate the binding of the immunoglobulin to host tissues or factors, including s cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).
The heavy chain of an antibody may or may not contain a terminal lysine (K), or a terminal glycine and lysine (GK). Thus, any of the heavy chain sequences and heavy chain constant region sequences ed herein can end in either GK or K, or lack K or GK, regardless of what the last amino acid of the sequence provides. This is because the al lysine and sometimes glycine and lysine are cleaved during sion of the antibody.
Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (KD) of 10-7 to 10-11 M or less. Any KD greater than about 10-6 M is generally ered to indicate binding nonspecifically. As used herein, an antibody that "binds specifically" to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means having a KD of 10-7 M or less, preferably 10-8 M or less, even more preferably x 10-9 M or less, and most preferably between 10-8 M and 10-10 M or less, but does not bind with high affinity to unrelated antigens. An antigen is "substantially identical" to a given antigen if it ts 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.
An immunoglobulin may be from any of the commonly known isotypes, including but not limited to IgA, ory IgA, IgG and IgM. The IgG isotype is divided in subclasses in some 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 are of the human IgG1 or IgG2 subtype. Immunoglobulins, e.g., human IgG1, exist in several allotypes, which differ from each other in at most a few amino acids. "Antibody" may include, by way of example, both naturally ing and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and nonhuman antibodies; wholly synthetic antibodies; and single chain dies.
The term "antigen-binding portion" of an dy or "antigen-binding fragment" of an antibody, as used herein, refers to one or more nts 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 within the term en-binding n" 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 linked by a disulfide bridge at the hinge ; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH s of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which ts of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded for by different genes, they can be linked, using inant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv ; see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci.
USA 85:5879-5883). Such 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 at Chan & Carter (2010) Nat. Rev. Immunol. :301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as intact dies. Antigen-binding ns can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
The term "amino acid sequence of conservative modifications form" refers to the amino acid modifications that do not significantly affect or alter the binding teristics of the antibody containing the amino acid sequence, and the modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid e is ed with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These es include amino acids with basic side chains (e.g., , arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, ine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid es from the same side chain family and the altered antibody can be tested for retained function using the functional assays described . Preferably, the vative modifications are no more than one or two in number.
Modification of the amino acid sequences described herein is desirable in the invention, especially those human heavy chain constant regions to adapt the sequence to the desired allotype, such as those found in Asian populations.
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 le. The ork regions can be human germline or non-germline gene sequences. In this way the framework can be germline, where one or more residues in the framework can be exchanged to match the residues in the most similar human germline ork at a comparable position. In this way, the binding member of the invention may be an isolated VH domain having a HCDR group within a human germline framework, for example, a human germline IgG VH framework. The binding member may also have a VL domain containing the LCDR group, such as in the human germline IgG VL framework.
The VH and/or VL scaffold residues can be modified as discussed, as ified herein, such as using site-directed mutagenesis. The VH or VL s, or binding members of the invention include such VL domains.
Changes can be made in one or more framework regions and/or one or more CDRs, the changes usually do not result in a loss of function, so a binding member comprising such changed amino acid sequence should maintain the ability to bind and/or neutralize LIF. It can maintain the same number of binding and/or neutralizing capabilities as the binding members that have not changed, as measured by the ical method described herein. A binding member comprising such changed amino acid sequence may have an improved ability to bind and/or lize LIF.
Changes can include the replacement of one or more amino acid residues with non-naturally occurring or andard amino acids, modifying one or more amino acid residues into a non-naturally ing or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acids into the ce.
Examples of the location and number of changes in the sequence of the ion are described elsewhere herein. Naturally occurring amino acids include the 20 "standard" L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard one-letter codes. 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 can be naturally occurring or non-naturally occurring. Several naturally occurring non-standard amino acids are known in the art, such as 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, N-ethylserine, etc. (Voet & Voet, 1995, Biochemistry, 2nd Edition, (Wiley)). Those amino acid residues derivatized at their N-α position will only be oned at the N-terminus of the amino acid sequence. Generally, the amino acid in the ion is an L-amino acid, but it may be a D-amino acid. Therefore changes may include modification with L-amino acids or replacement of o acids with D-amino acids. The formylated, acetylated and/or phosphorylated forms of amino acids are known, and the amino acids of the ion can be modified as such.
The amino acid ces in the binding members and antibody domains of the ion may include the unnatural or non-standard amino acids described above. andard amino acids (such as D-amino acids) can be incorporated into the amino acid sequence during synthesis, or by modification or substitution of "original" rd amino acids after amino acid synthesis.
The use of non-standard and/or non-naturally occurring amino acids improves the diversity of structure and function, and can increase the potential to achieve the desired LIF binding and neutralizing properties in the binding members of the invention. In addition, compared with standard L-amino acids, D-amino acids and their analogs have been shown to have better pharmacokinetic properties due to the degradation of polypeptides with L-amino acids in vivo after administration to animals such as humans.
The generation of the new VH or VL region with CDR-derived sequences of the invention can use one or more random mutagenesis ed from VH and/or VL genes to generate mutants in all the variant regions. Such technique is 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 in all variant regions or CDR groups.
Another method that can be used is targeted mutagenesis of the CDR regions of VH or VL genes. Such method is published by Barbas et al. s 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).
All the s bed above are known in the art, and those skilled in the art will be able to use such methods and adopt conventional methods in the art to provide binding members of the invention.
Any VH and VL domain amino acid sequence ts with the specific sequences disclosed herein can be used in accordance with the invention, as sed.
Specific variants may e one or more amino acid sequence changes (additions, deletions, substitutions and/or insertions of amino acid residues). In some embodiments, the t has less than about 17, less than 9, or less than 5 such changes.
As shown above, the CDR amino acid sequence substantially as described herein can be d as a CDR in a human antibody variant structure region or most of it.
The HCDR3 sequence substantially as described herein represents an embodiment of the invention, each of these can be carried as a CDR in a human antibody variant region or most of it, optionally in combination with HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3 of the invention.
The term "monoclonal dy," as used herein, refers to an antibody that displays a single binding specificity and affinity for a specific epitope or a composition of dies in which all antibodies display a single binding specificity and affinity for a specific epitope. lly such monoclonal antibodies will be derived from a single antibody encoding cell or nucleic acid, and will be propagated without intentionally introducing any sequence alterations. Accordingly, the term "human monoclonal antibody" refers to a monoclonal antibody that has variable and optional constant regions derived from human germline immunoglobulin sequences.
In one embodiment, human monoclonal antibodies are produced by a hybridoma, for example, ed by fusing a B cell derived 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), with an immortalized cell. The term "mAb" refers to monoclonal antibodies.
The term "recombinant human antibody," as used herein, includes all human antibodies that are prepared, expressed, produced or isolated by recombinant means, such as (a) dies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody y, and (d) dies prepared, expressed, produced or isolated by any other means that e splicing of human immunoglobulin gene sequences to other DNA sequences. Such inant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences and are encoded by the germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody tion. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117- 1125), the variable region ns the antigen binding domain, which is encoded by various genes that nge to form an antibody specific for a exogenous antigen. In addition to rearrangement, the variable region can be further modified by le single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody to the exogenous antigen. The constant region will change in further response to an n (i.e., isotype switch). Therefore, the rearranged and somatically mutated nucleic acid ces that encode the light chain and heavy chain immunoglobulin polypeptides in se to an antigen may not be identical to the original germline sequences, but instead will be substantially identical or similar (i.e. , have at least 80% identity).
A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin ces. Furthermore, if the antibody contains a constant , the constant region is also derived from human ne immunoglobulin sequences.
The antibodies described herein may include amino acid residues not d by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by c mutation in vivo). However, the term "human antibody", as used herein, is not ed to include antibodies in which CDR sequences d from the germline of another mammalian s, such as a mouse, have been grafted onto human ork sequences. The terms "human" antibodies and "fully human" antibodies are used synonymously.
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 with corresponding amino acids derived from human immunoglobulins. In one embodiment of an antibody in humanized form, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate 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.
A "chimeric dy" refers to an antibody in which the variable regions are derived from one species and the nt regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse dy and the constant regions are derived from a human antibody.
The functional antibody fragments of the invention include any functional fragments whose half-life period is increased by chemical modification such as by PEGylation or incorporation into liposomes.
The antibodies of the invention e bispecific antibodies. Bispecific or bifunctional antibodies form second-generation onal antibodies, in which two different variant regions are combined into the same molecule (Holliger and Bohlen, 1999 Cancer and metastasis rev. 18: 411-419). For their ability to recruit new effector functions or to target some molecules on the surface of tumor cells, their applications in the field of diagnosis and treatment have been elucidated. When bispecific antibodies are used, for example, omas that are chemically prepared or derived from , can be conventional bispecific antibodies, which can be manufactured in various ways (HolligerP. & Winter G. Current Opinion Biotechnol. 4, 446-449: 1993), or can be any of the bispecific antibody fragments mentioned above. These antibodies can be ed by al methods or somatic methods, but equally and preferably c engineering methods, which allow heterodimerization to be carried out, and facilitates the purification process of the obtained antibodies. Examples of bispecific antibodies include those of the BiTETM method, in which the binding domains of two antibodies with different specificities can be used and linked directly by a short flexible peptide. This combines two antibodies on a short single polypeptide chain.
The diabody and scFc are constructed without the Fc region, only variant region is used, which potentially reducing the effect of the anti-idiotypic response.
Bispecific antibodies can be constructed as full IgG, ific (Fab')2, (Fab')PEG, y or other bispecific scFv. rmore, two bispecific antibodies can be linked to form a tetravalent antibody using conventional methods known in the Compared with bispecific whole antibodies, ific diabodies are also particularly useful because they can be easily constructed and sed in E. coli.
Using a phage display library (WO1994/13804), diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificity can be easily ed. If one arm of the diabody is kept constant, then a y is prepared, in which the other arms are mutated, and antibodies of appropriate specificity are selected. Bispecific whole antibodies can be prepared by different engineering methods, which are bed in Ridgeway et al. way, J.B.B. et al., Protein Eng. 9, 616-621, 1996) or WO1996/27011, WO1998/50431 and WO2006/028936.
A "modified heavy chain constant region" refers to a heavy chain constant region comprising the constant domains CH1, hinge, CH2, and CH3, wherein one or more of the constant domains are from a different isotype (e.g. IgG1, IgG2, IgG3, IgG4). In some embodiments, the modified constant region includes a human IgG2 CH1 domain and a human IgG2 hinge fused to a human IgG1 CH2 domain and a human IgG1 CH3 domain. In certain ments, such modified constant regions also include amino acid modifications within one or more of the domains relative to the wildtype amino acid sequence.
As used herein, pe" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that is d by the heavy chain constant region genes.
"Allotype" refers to naturally occurring variants in a specific isotype group, which variants differ in a few amino acids (see, e.g., Jefferis et al. (2009) mAbs 1: 1).
Antibodies described herein may be of any allotype.
Unless specified otherwise herein, all amino acid s are according to the EU index of the Kabat system , E. A., et al. (1991) Sequences of ns of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
The terms "an dy recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen." The term "an isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to LIF is substantially free of antibodies that specifically bind antigens other than LIF). An ed antibody that specifically binds to an epitope of LIF may, however, have cross-reactivity to other LIF proteins from different species.
An "effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom. Exemplary tor functions" include C1q g, complement dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector ons such as ADCC and antibody dependent ediated hagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR). Such effector ons generally require the Fc region to be combined with a binding domain (e.g., an antibody variable ).
An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody comprise receptors of the FcγR family, including allelic variants and alternatively spliced forms of these receptors.
The FcγR family ts of three activating receptors (FcγRⅠ, FcγRⅢ, and FcγRⅣ in mice; FcγRⅠA, FcγRⅡA, and FcγRⅢA in ) and one inhibitory receptor (FcγRⅡB). Various properties of human FcγRs are summarized in Table A. The majority of innate effector cell types coexpress one or more activating FcγR and the tory FcγRⅡB, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcγRⅢ in mice and FcγRⅢA in humans) but does not express the inhibitory FcγRⅡB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered that the types of activating Fc receptors which it binds to are equivalent to murine IgG2a.
Table A. Properties of human FcγRs Fcγ Allelic Affinity for Isotype Cellular bution variants human IgG preference FcγRⅠ None High（KD IgG1=3>4>>2 Monocytes, macrophages, described ~10nM） activated neutrophils, dentritic cells FcγRⅡA H131 Low to IgG1>3>2>4 Neutrophils, monocytes, medium macrophages, eosinophils, R131 Low IgG1>3>4>2 dentritic cells, platelets FcγRⅢA V158 Medium >>4>2 NK cell, monocytes, macrophages, mast cells, F158 Low IgG1=3>>4>2 eosinophils, dentritic cell FcγRⅡB I232 Low IgG1=3=4>2 B cells, monocytes, macrophages, dentritic cells, T232 Low IgG1=3=4>2 mast cells A "hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to the domain of a heavy chain constant region that links the CH1 domain to the CH2 domain and includes the upper, middle, and lower portions of the hinge (Roux et al. J.
Immunol. 1998 161:4083). The hinge provides varying levels of ility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions.
The term " includes wildtype hinges, as well as variants thereof (e.g., non-naturally-occurring hinges or modified ). For example, the term "IgG2 hinge" includes wildtype IgG2 hinge, 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.
The term "CH1 domain" refers to the heavy chain constant region linking the variable domain to the hinge in a heavy chain constant domain. The term "CH1 domain" includes wildtype CH1 domains, as well as ts thereof (e.g., non-naturally-occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes pe CH1 s 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 CH1 domains include CH1 domains with ons that change a biological activity of an antibody, such as ADCC, CDC or half-life period.
The term "CH2 domain" refers to the heavy chain constant region g the hinge in a heavy chain constant domain to the CH3 domain. The term "CH2 domain" es wildtype CH2 domains, as well as variants thereof (e.g., non-naturally-occurring CH2 domains or modified CH2 domains). For example, the term "CH2 " es wildtype CH2 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 CH2 domains include CH2 domains with mutations that change a biological activity of an antibody, such as ADCC, CDC or half-life period.
The term "CH3 domain" refers to the heavy chain nt region that is C-terminal to the CH2 domain in a heavy chain constant domain. The term "CH3 domain" includes wildtype CH3 domains, as well as variants thereof (e.g., non-naturally-occurring CH3 domains or ed CH3 domains). For example, the term "CH3 domain" includes wildtype 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 a biological ty of an antibody, such as ADCC, CDC or half-life period.
A "CL domain" refers to the constant domain of a light chain. The term "CL domain" includes wildtype CL domains and variants thereof.
A "native sequence Fc region" or e sequence Fc" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature.
Native sequence human Fc regions include a native sequence human IgGl Fc region; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants f.
Native sequence Fc includes the various allotypes of Fcs (see, e.g., Jefferis et al. (2009) mAbs 1: 1).
The term "epitope" or "antigenic determinant" refers to a site on an n (e.g., LIF) to which an immunoglobulin or antibody ically binds. Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear e) or noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not , retained when exposing to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost when treating with denaturing solvents. An e typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or amino acids in a unique spatial conformation. Methods for determining what es are bound by a given dy (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation is, wherein overlapping or contiguous peptides (e.g., from LIF) are tested for reactivity with a given antibody (e.g., anti-LIF dy). Methods of ining spatial conformation of epitopes include techniques in the art and those described , for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular y, Vol. 66, G. E. , Ed. (1996)).
Antibodies that "compete with another antibody for binding to a target" refer to antibodies that inhibit (partially or completely inhibit) the binding of another antibody to the target. Whether the two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of another antibody to a target, may be ined using known competition experiments, such as those described in the Examples. In certain embodiments, an antibody competes with another antibody, and inhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the binding. The extent of tion or competition may be different depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is incubated first with the target). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Pro toe ; 2006; doi: 10.1101/pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor tory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, the overlapping epitope or to the adjacent epitopes (e.g., as evidenced by steric hindrance).
Other itive 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. 14 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich analysis (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using I-125 label (see Morel et al., Mol.
Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J.
Immunol. 32:77 (1990)).
The term "Kassoc" or "Ka", as used herein, is intended to refer to the association rate constant of a specific dy-antigen interaction, whereas the term "Kdis" or "Kd" as used herein, is intended to refer to the dissociation rate constant of a specific antibody-antigen ction. The term "KD", as used herein, is intended to refer to the equilibrium dissociation constant, which is ed from the ratio of Kd to Ka (i.e,.
Kd/Ka) and is expressed as a molar concentration (M). KD values of antibodies can be determined using s well established in the art. A preferred method for determining the KD of an antibody is to analyze by using surface plasmon resonance, preferably using a biosensor system such as a e® e plasmon resonance system or flow cytometry and Scatchard.
The term "EC50" in the context of an in vitro or in vivo assay using an antibody or antigen binding fragment thereof, refers to the concentration of an antibody or an antigen-binding portion thereof that induces a response that is 50% of the maximal response, i.e., y n the l response and the baseline.
The term "IC50", in functional analysis, IC50 is the concentration of the binding member that can reduce the biological response to 50% of its maximum value, taking nM as the unit. In -binding studies, IC50 is the concentration that reduces receptor binding to 50% of the maximum specific binding level. The IC50 can be calculated by plotting the percentage of the m biological activity response as a function of the log of the binding member concentration, and using a software program such as Origin (OriginLab Software Company, Northampton, Massachusetts, USA) to fit the S function to the data to generate the IC50 value. The potency is determined or measured using one or more analytical methods known to those skilled in the art and/or described or referenced herein. The neutralizing potency of the binding members can be sed as the n.
The term "naturally-occurring" as used herein as d to an object refers to the fact that an object can be found in nature. For example, a ptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein may n a modification such as, but not limited to, glycosylation, phosphorylation or a disulfide bond. A "protein" may comprise one or more polypeptides.
The term ic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. A c acid molecule may be a single chain or a double chain, and may be cDNA. Also provided are rvative sequence modifications" of the sequences set forth in SEQ ID NOs described herein, i.e., nucleotide and amino acid sequence modifications which do not abrogate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid ce, to the antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as, nucleotide and amino acid additions and deletions. For example, cations can be introduced into SEQ ID NOs described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions, in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, cine, proline, phenylalanine, nine), beta-branched side chains (e.g., ine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
For nucleic acids, the term "substantial identity" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are cal, with appropriate nucleotide insertions or deletions, 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. Alternatively, substantial identity exists when the segments will hybridize under selective hybridization conditions, to the complement of the chain.
For polypeptides, the term "substantial ty" indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the amino acids.
The identity% between two ces is a function of the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., identity% = number of identical ons/total number of positions x 100), with optimal alignment determined taking into account the number of gaps, and the length of each gap, which need to be uced for optimal alignment of the two ces.
The comparison of sequences and ination of percent identity between two sequences can be accomplished using a mathematical thm, as described in the non-limiting examples below.
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 a 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 algorithm of E. Meyers and W.
Miller (CABIOS, 4: 11-17 (1989)) which has been 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 algorithm of Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 , 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.
The nucleic acid and protein ces described herein can further be used as a "query ce" to perform searches against public databases to, for e, identify related sequences. Such searches can be performed with 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 with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences identical to the nucleic acid les described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences identical to the protein molecules described herein. To obtain gapped alignments for ison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) c Acids Res. :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.
These nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. The nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular c acids (e.g., the other parts of the chromosome) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).
Nucleic acids, e.g., cDNA, may be mutated, in accordance with standard techniques to provide gene ces. For encoding sequences, these mutations may affect amino acid sequence as desired. ically, DNA sequences substantially identical to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated.
The term "vector," as used herein, is intended to refer to a nucleic acid le capable of transporting another nucleic acid to which it has been linked. One type of vector is "plasmid," which refers to a circular double chains DNA loop into which other DNA segments may be linked. Another type of vector is a viral vector, n other DNA segments may be linked into the viral genome. Some vectors are capable of autonomous ation in a host cell into which they are uced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
Other s (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell, and thereby are replicated along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant sion vectors" (or simply, "expression vectors"). In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. In the present description, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, also included are other forms of expression s, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term "recombinant host cell" (or simply "host cell"), as used herein, is ed to refer to a cell that comprises a nucleic acid that is not lly present in the cell, and maybe a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific subject cell but to the progeny of such a cell. Since certain modifications may occur in succeeding tions due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.
As used herein, the term "antigen" refers to any l or synthetic immunogenic substance, such as a protein, peptide, or hapten. An n may be LIF or a fragment f.
As used herein, the terms "inhibition" or "blocking" (e.g., referring to inhibition/blocking of LIF binding or activity) are used hangeably and encompass both partial and complete inhibition/blocking.
As used herein, "cancer" refers a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Since unregulated cell division may result in the formation of malignant tumors or cells, they would invade neighboring tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.
The terms "treat," ing," and "treatment," as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of ing, alleviating, ameliorating, inhibiting, or slowing down or ting the progression, development, severity or recurrence of a symptom, complication, condition or mical indicia associated with a disease. Prophylaxis refers to administration to a t who does not have a disease, to prevent the disease from occurring or minimize its effects if it does.
The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least lly achieve a desired effect. A "therapeutically ive amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another eutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. A "prophylactically effective amount" or a "prophylactically effective dosage" 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 developing a disease or of suffering from a recurrence of 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 the disease can be evaluated using a variety of s known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in , or by assaying the activity of the agent in in-vitro assays.
The terms "patient" and "subject" refer to any human or non-human animal that receives either prophylactic or eutic treatment. For e, the methods and compositions described herein can be used to treat a subject having cancer. The term "non-human " includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
Examples Example 1 Screening and Identification of Anti-Human LIF Monoclonal Antibody 1.1 Preparation of anti-human LIF onal antibody 38E10E1C11 by hybridoma method ing to the monoclonal antibody preparation method (Kohler and Milstein (1975) Nature 256: 495), recombinant human LIF protein (purchased from Sino Biological) was used for immunizing BABL/c mice. 25μg recombinant human LIF protein with an equal volume of Freund's te adjuvant was used for the l immunization by multiple aneous injections of the back. After four weeks, Freund's incomplete adjuvant plus 25μg of recombinant human LIF protein was used for the second immunization. Indirect ELISA method was used to detect the titer of antibody20 days later. After 2-3 weeks interval, 50μg of recombinant human LIF protein was injected intraperitoneally to strengthen the immunization. After 3 days, the animals were sacrificed and spleen cells were taken for fusion.
The mouse myeloma cells SP2/0 in logarithmic growth phase were taken for counting, and the spleen cell suspension of the immunized mouse was prepared. The spleen cells were fused with SP2/0 cells using 50% PEG according to conventional methods. The fused cells were added to a 96-well plate of trophoblast cells (six-week-old BABL/c mouse peritoneal macrophages), and were screened and cultured in DMEM with 1% HAT and 20% fetal bovine serum. When the clone was grown to 1/3 of the bottom of the plate, the culture supernatant was collected. ELISA plates were coated by recombinant human LIF protein, and indirect ELISA method was used for detecting of anti-LIF antibody in culture supernatant and screening clones secreting uman LIF antibody. rmore, a cell line stably secreting a high affinitive anti-human LIF monoclonal antibody was obtained by monoclonal dyization using limiting on, and the secreting antibody is labeled as 38E10E1C11. The full-length gene sequences encoding the light and heavy chains of the 38E10E1C11 antibody were determined as shown in SEQ ID NO:42 and SEQ ID NO:44, respectively, and the corresponding full-length amino acid ces of the light and heavy chains of the 38E10E1C11 antibody were shown in SEQ ID NO:41 and SEQ ID NO:43, respectively; the gene sequences encoding the le region of the light and heavy chains of the 38E10E1C11 antibody were shown in SEQ ID NO:76 and SEQ ID NO:77, respectively; and the ponding amino acid sequences of the variable regions of the light and heavy chains of the 38E10E1C11 antibody are shown in SEQ ID NO:74 and SEQ ID NO:75. According to the Kabat system, the amino acid sequence of LCDR1 of 1C11 antibody was shown in SEQ ID NO:1, the amino acid sequence of LCDR2 was shown in SEQ ID NO:2, the amino acid sequence of LCDR3 was shown in SEQ ID NO:3, the amino acid sequence of HCDR1 was shown in SEQ ID NO:4, and the amino acid sequence of HCDR2 was shown in SEQ ID NO:45, and the amino acid sequence of HCDR3 was shown in SEQ ID NO:6. The immunoglobulin type and subtype of the 1C11 antibody were identified (the result is IgG1 subtype, κ type light chain).
After obtaining a hybridoma cell line capable of stably secreting antibodies, the cell was domesticated by the CD Hybridoma serum-free medium of thermo fisher and adapted to the serum-free suspension shake culture, and then the antibody was expressed and ed using the serum-free medium. 1.2 Preparation of anti-human LIF monoclonal antibody P36-033 by phage display technology Recombinant human LIF n was used for immunizing BABL/c mice. 25μg recombinant human LIF protein with an equal volume of Freund's complete adjuvant was used for the initial immunization by le subcutaneous injections of the back.
After four weeks, Freund's incomplete adjuvant plus 25μg of recombinant human LIF protein is used for the second immunization. Indirect ELISA method was used to detect the titer of antibody 20 days later. After 2-3 weeks al, 50μg of recombinant human LIF protein was injected eritoneally to strengthen the immunization. After 3 days, the animals were sacrificed and spleen cells were taken for fusion. Total RNA from spleen cells was extracted using TRIZOL Reagent from Invitrogen y and reverse ribed to cDNA using ogen cDNA Reverse Transcription Kit. The antibody gene was amplified by the degenerate primers of mouse light and heavy chain le region and constructed into a phage display vector, and a phage antibody library was constructed. Thermo-automatic magnetic bead sorting system was used to elimination and selection of the phage antibody library, and the phage ELISA was used to select an E. coli clone e of binding the recombinant human LIF protein, and the sequence of the antibody was determined. Furthermore, the P36-033 antibody was obtained by ELISA and cell viability identification. The full-length gene sequences of the P36-033 light chain and heavy chain 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 gene sequence encoding the variable region of 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 region of the light and heavy chains of the P36-033 antibody are shown in SEQ ID NO: 82 and SEQ ID NO:83.
According to Kabat system, 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, the amino acid sequence of HCDR1 is shown in SEQ ID NO:69, the amino acid sequence of HCDR2 is shown in SEQ ID NO:70, the amino acid sequence of HCDR3 is shown in SEQ ID NO:71. 1.3 Expression and purification of ve l antibody 5D8 According to the report of the patent document of antibody 5D8 is an antibody that inhibits the g of LIF protein and GP130.
According to the patent document, le gene sequences of the invention were synthesized, and different light and heavy chains are paired in ent combinations to construct the full-length antibodies in the form of Human IgG1, and one of them with the best binding to LIF protein was found eventually, and meanwhile it was capable of blocking the recombinant human LIF protein binding with human GP130 and blocking the STAT3’s phosphorylation in HCT116 cells by the recombinant human LIF protein through cell viability identification, therefore, the invention named it 5D8 as a positive control antibody in a follow-up trial. The full-length gene sequences encoding the heavy chain and light chain of the 5D8 antibody were determined as shown in SEQ ID NO:63 and SEQ ID NO:65, tively, and the corresponding full-length amino acid sequences of the heavy chain and light chain of the 5D8 antibody were shown in SEQ ID NO:62 and SEQ ID NO:64, respectively; the gene sequences encoding the variable region of the 5D8 antibody were determined as shown in SEQ ID NO:80 and SEQ ID NO:81, respectively, and the corresponding amino acid sequences of the variable region of the heavy chain and the light chain of the 5D8 antibody are shown in SEQ ID NO:78 and SEQ ID NO:79, tively.
Example 2 The Binding Experiment of the Anti-Human LIF Antibody to Human LIF Protein The recombinant human LIF protein was diluted to 1μg/mL, was coated on the enzyme label plate,100μL of the n was added into every well, and incubated ght in 4 °C. The enzyme label plates were taken out next day, and the liquid was discarded, and were washed with PBST three times and blocked with 2% BSA in PBS for 1h at room temperature, then washed with PBST three times, and added anti-human LIF dies 38E10E1C11, 5D8 and P36-033 at different concentrations and incubated for 1h at room temperature. The liquid was discarded, and the plates were washed with PBST four times. The plates were added with HRP-labeled goat anti-mouse Fab antibody or goat anti-human FC antibody and incubated for 1h at room temperature. Then the liquid was discarded, and the plates were washed four times with PBST and incubated with TMB colored solution in 10 minutes at room temperature. Stop color development by adding 2 mol/L H2SO4, and the absorption value at 450 nm is quantitated using an ted plate photometer, and the results were shown in Figure 1. The results show that the binding of the 38E10E1C11 antibody and the P36-033 antibody to human LIF n is stronger than the binding of the 5D8 antibody to human LIF protein.
Example 3 The Binding Experiment of the Anti-Human LIF Antibody to Mouse LIF Protein The recombinant mouse LIF protein (purchased from ACRO Biosystems) was diluted to 1μg/mL, and was coated on the enzyme label plate, 100μL of protein was added into every well, and incubated overnight in 4 °C. The enzyme label plates were taken out next day, and the liquid was discarded, and were washed with PBST three times and blocked with 2% BSA in PBS for 1h at room ature, then washed with PBST three times, and added uman LIF antibodies 38E10E1C11, 5D8 and P36-033 at different concentrations and incubated for 1h at room temperature. The liquid was discarded, and the plates were washed with PBST four times. The plates were added with HRP-labeled goat anti-mouse Fab antibody or goat uman FC antibody and incubated for 1h at room temperature. Then the liquid was discarded, and the plates were washed four times with PBST and incubated with TMB colored solution in 10 minutes at room temperature. Stop color development by adding 2 mol/L H2SO4, and the absorption value at 450 nm is quantitated using an automated plate eter, and the results were shown in Figure 2. The results show that the 38E10E1C11 antibody has no binding activity to the mouse LIF protein, and the P36-033 antibody has binding ty to the mouse LIF protein which is less than the 5D8 antibody. e 4 The Binding Experiment of the Anti-Human LIF Antibody to Machin LIF Protein The recombinant machin LIF protein (purchased from Sino biological) was diluted to 0.5μg/mL, and was coated on the enzyme label plate, 100μL of protein was added into every well, and ted overnight in 4 °C. The enzyme label plates were taken out next day, and the liquid was discarded, and were washed with PBST three times and blocked with 2% BSA in PBS for 1h at room temperature, then washed with PBST three times, and added anti-human LIF antibodies 38E10E1C11, 5D8 and P36-033 at different trations and incubated for 1h at room temperature. The liquid was discarded, and the plates were washed with PBST four times. The plates were added with beled goat anti-mouse Fab antibody or goat anti-human FC antibody and incubated for 1h at room temperature. Then the liquid was discarded, and the plates were washed with PBST four times and incubated with TMB colored solution in 10 s at room temperature. Stop color development by adding 2 mol/L H2SO4, and the absorption value at 450 nm is quantitated using an automated plate photometer, and the results were shown in Figure 3. The results show that the binding activities of the 38E10E1C11 and the P36-033 are better than the binding activity of the 5D8 .
Example 5 The Affinity Assay of the Anti-Human LIF antibodies The affinity of the purified monoclonal antibodies to the recombinant human LIF protein was determined by KinExA 4000. 200 mg PMMA hard beads were added with 30μg of the 38E10E1C11 antibody and additional adding coating solution to 1mL. The buffer composition is 1×PBS, pH 7.4, 0.02% NaN3. And make sure that the beads were completely suspended in the solution, and rotated for 2h at room temperature. The beads were naturally settled or quickly centrifuged at low speed.
The supernatant was removed and the beads were blocked with PBS ning 1% BSA. 15 mL of 300 pM antigen solution and 15 mL of 240 pM Ab2 (38E10E1C11) solution were prepared. 0.6 mL of 300 pM antigen and 0.6 mL of 240 pM antibody Ab2 E1C11) were put into different sample tubes separately. The samples in the two tubes were well mixed and put together into one tube, the concentration of the n was150pM and the tration of antibody Ab2 (38E10E1C11) was 120 pM at this time, and the solution was placed in the corresponding position in the tube holder. 16 groups were prepared, and the incubation time of each group was different.
Each group was added with 1μg/Ll Streptavidin Protein, t 650 Solution, and was detected on machine set to incubate for 24 hours. In the KinExatm Pro software, the equilibrium dissociation constant (Kd) for n-curve analysis was calculated by the unknown ligand model, and the results are shown in Figure 4. The results show that the affinity of the 38E10E1C11 mAb to human LIF protein is 4.52x10-12M.
Example 6 The P36-033 mAB and the 38E10E1C11 mAb compete with LIFR for binding to human LIF protein The recombinant human LIF protein was coated on the enzyme label plate at a tration of 1μg/mL, LIFR protein (expressed y with human FC) at a concentration of 0.6125μg/mL was added (50μL/well), and the anti-human LIF antibodies 38E10E1C11, P36-033 and 38E10E1C11R which is inantly expressed by CHO cells (SEQ ID NOs: 41 and 43) at different concentrations simultaneously were added separately (50μL/well) and incubated for 2h at room temperature. After washed four times with PBST, incubated with HRP-labeled goat anti-mouse FC secondary antibody for 1h at room temperature. Then the plates were washed four times with PBST and added with TMB coloured solution in for 10 minutes. The absorption value at 450 nm is quantitated using an -labelling measuring instrument, and the data was analyzed and d using Origin pro 9 software. The results were shown in Figure 5. The results show that the 38E10E1C11 mAb and 38E10E1C11 can inhibit the binding of human LIF to human LIFR, while the P36-033 mAb can’t inhibit the binding of human LIF to human LIFR.
Example 7 The P36-033 mAB and the 38E10E1C11 mAb e with GP130 for binding to human LIF protein The recombinant human LIF protein was coated on the enzyme label plates at a concentration of 1μg/mL, GP130 protein ssed fusedly with human FC) at a concentration of L was added well), and the anti-human LIF antibodies P36-033 and 38E10E1C11 at different concentrations simultaneously were added separately (50μL/well), and incubated for 2h at room temperature. At the same time, the control wells added with antibodies and without GP130 protein were set.
Incubated for 2h at room temperature. After washed four times with PBST, ted with HRP-labeled goat anti-mouse FC secondary antibody for 1h at room temperature.
Then the plates were washed four times with PBST and added with TMB coloured solutionin for 10 minutes. The absorption value at 450 nm is quantitated using an enzyme-labelling measuring instrument, and the data was analyzed and plotted using Origin pro 9 software. The results were shown in Figure 6. The s show that the P36-033 and the 38E10E1C11 can inhibit the g of human LIF to human GP130 Example 8 Detection of the specificity of 38E10E1C11 mAb The human LIF, human IL-6 , human OSM , human CNTF (purchased from Sino biological) were coated on the enzyme label plate at a concentration of 1μg/mL separately, and the anti-human LIF antibodies 38E10E1C11, P36-033 and 5D8 at different concentrations were added separately and incubated for 1h at room temperature. Then the plates were washed four times with PBST and incubated with HRP-labeled goat anti-mouse FC secondary antibody for 1h at room ature.
Then the plates were washed four times with PBST and added with TMB colored solution for color development at room temperature for 10 minutes. The absorption value at 450 nm is quantitated by the enzyme-labeling measuring instrument, and the data was analyzed and plotted using Origin pro 9 software. The results were shown in Figure 7. The results show that the 38E10E1C11 mAb and the P36-033 mAb only bound to human LIF protein and didn’t bind to human IL-6, human OSM and human CNTF, while the 5D8 antibody bound to human OSM and human CNTF n.
Example 9 The western blot detection that 38E10E1C11 mAb can be used for human LIF protein The recombinant human LIF protein was d to the concentration shown in Figure 8,. the cell supernatant of CT26 and C26-hLIF cells cultured for three days with 5XSDS-PAGE g buffer was boiled for 10 minutes. 10μL sample was taken for SDS-PAGE electrophoresis, and then the electrophoresis band was transferred to PVDF membrane for western blot detection, the y antibody used for detection is 38E10E1C11 antibody at the concentration of 1μg/mL, incubated for 2h at room temperature, and washed three times by TBST buffer. Then adding 1:3000 diluted HRP-labeled goat anti-mouse secondary antibody, and incubated for 2h at room temperature, incubated for 2h at room temperature, and washed three times by TBST buffer. Then incubated with enhanced uminescence solution (Perice), and detected and photographed by the Amersham Imager 600 ultra-sensitive multi-function imager. The results were shown in Figure 8. The results show that the 38E10E1C11 mAb can be used for western blot detection of human LIF protein.
Example 10 Anti-human LIF antibody cell activity assay .1 Detection of Inhibiting STAT3 Activation in HCT116 cells HCT116 cells were digested and centrifuged, then the cells were ended and plated at 12-well plate in a volume of 1mL with 5x105 cells/well. Then the cells were ted at 37 °C, 5% CO2 overnight. The original medium was discarded next day, and the cell culture medium containing 100ng/mL of inant human LIF protein and anti-LIF antibodies in different concentrations were added and ted for 30 minutes at 37 °C, and at the same time the control wells containing no recombinant human LIF protein and only recombinant human LIF protein without antibodies were set. The medium was then removed and 100μL 1x lysate was added to each well of the l plate, and the cells were lysed on ice for 30 min. The lysate was transferred to a 1.5 mL centrifuge tube, and the tube with lysate was fuged at 13,000 rpm for 10 min, and the supernatant was collected. The supernatant was taken for western blot detection of the phosphorylation of STAT3.
The results were shown in Subfigure A in Figure 9 and Figure 11. From the results, it was revealed that the 1C11 antibody and the P36-033 antibody can inhibit the phosphorylation of STAT3 in HCT116 cells induced by human LIF protein. .2 Detection of the activity of the Anti-Human LIF antibodies h the test of inhibiting STAT3 activation in KP4 cells KP4 cells were digested and centrifuged, and the cells were resuspended and plate at 12-well plate in a volume of 1mL with 5x105 cells/well. Then the cells were incubated at 37 °C, 5% CO2 overnight. The original medium was ded next day, and the cell culture medium containing 50ng/mL of recombinant human LIF protein and anti-LIF dies in different concentrations were added and incubated for 30 minutes at 37 °C, and at the same time the control wells containing no recombinant human LIF protein and only recombinant human LIF protein without antibodies were set. The medium was then removed and 100μL 1x lysate was added to each well of the 12-well plate, and the cells were lysed on ice for 30 min. The lysate was transferred to a 1.5 mL centrifuge tube, and the tube with lysate was centrifuged at 13,000 rpm for 10 min, and the supernatant was collected. The supernatant was taken for western blot ion of the phosphorylation of STAT3. The results were shown in Subfigure B in Figure 9, it shows that the 38E10E1C11 antibody can inhibit the phosphorylation of STAT3 in KP4 cells induced by human LIF protein. .3 Detection of the ty of the Anti-Human LIF antibodies through the test of inhibiting STAT3 activation in KP4 cells KP4 cells were digested and fuged, and the cells were resuspended and plate at 12-well plate in a volume of 1mL with 5x105 cells/well. Then the cells were incubated at 37 °C, 5% CO2 overnight. The original medium was ded next day, and the anti-LIF antibodies in different concentrations and the cell culture medium of CT26-hLIF cells in a volume ratio of 1:1 was added and incubated for 30 minutes at 37 °C, and at the same time the control wells containing the culture supernatant of CT26 were set. The medium was then removed and 100μL 1x lysate was added into the cells, and the cells were lysed on ice for 30 min. The lysate was transferred to a 1.5 mL fuge tube, and the tube with lysate was centrifuged at 13,000 rpm for 10 min, and the atant was collected. The supernatant was taken for western blot detection of the phosphorylation of STAT3. Figure 10 shows that the 1C11 antibody can inhibit the phosphorylation of STAT3 in KP4 cells induced by human LIF protein secreted by the CT26-hLIF cells. .4 Detection of LIF dy activity through the test of M1 cell proliferation M1 cells were centrifuged and washed twice by RPMI1640 , the M1 cells were plated into 96-well plate in a volume of 100μL cell per well at a density of 2x105cells/mL. The cell culture medium containing 10ng/mL of recombinant human LIF protein and anti-LIF antibodies in different concentrations were added until the volume of every well reached 200μL finally and incubated for 72h at 37 °C in the tor. At the same time, the control wells without human LIF protein were set.
CCK-8 was added to measure cell proliferation. The results were shown in Figure 12, and the results show that both the 10E1C11 mAb and the P36-033 mAb can reverse the inhibition induced by human LIF protein of the proliferation of M1 cells, .
Example 11 Detection of Anti-tumor activity of anti-human LIF antibody in The 1C11 antibody did not cross-react with the mouse LIF protein by ELISA assay. In order to carry out the activity evaluation in vivo, CT26 cell line overexpressing human LIF protein needed to be constructed. According to the literature, human LIF protein is able to bind to the LIFR and GP130 on the e of mouse cells, thereby activating the downstream signal. Therefore, it was speculated that human LIF protein secreted by CT26 cells which expressed human LIF protein highly can inhibit the immune system of mice, and the anti LIF protein can release the inhibitory effect and thus exert an anti-tumor effect. 11.1 Establishment of CT26 cell line overexpressing human LIF - The mouse colon cancer cell line CT26 was infected with the constructed lentivirus containing human LIF gene. The expression of LIF protein was detected after 48h. The cell line was cloned by ng dilution method, and the medium with puromycin in the final tration of 1μg/mL was added for pressurized screening.
Finally CT26 cell line stably and highly expressing human LIF n was obtained. 11.2 Anti-tumor activity of uman LIF antibody detected by CT26-hLIF BABL/C subcutaneous implantation model CT26-hLIF cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, collecting the cells in the logarithmic growth phase, resuspended in PBS to 107 cells/mL, and inoculate BABL/c mice subcutaneously. One day after the inoculation, the mice were divided into groups and injected with vehicle l, anti-human LIF antibody respectively, the administration concentration is 15mg/kg body weight, twice a week, for 4 consecutive weeks, the tumor volume is measured twice a week, the tumor growth curve is drawn, and the tumor inhibition rate is calculated. The results were shown in Figure 13. The results show that the 38E10E1C11 mAb can inhibit CT26-hLIF cells proliferate in BABL/c mice. 11.3 Measurement of the sensitivity of different atic cancer cell lines to the stimulation of LIF protein The human pancreatic cancer cell lines Panc02.03, KP4, MIA paca2 were inoculated into 6-well plate with a density of 106cell/well separately. The medium was ed with the fresh medium after overnight incubated, and 50ng/mL inant human LIF protein and the 38E10E1C11 antibody were added, meanwhile the control wells without LIF protein were set. The ent wells and the control wells were incubated at 37 °C for 30 minutes. The medium was then removed and 200μL 1x lysate was added to the cells of 200μL for each well, and the cells were lysed on ice for 30 min. The lysate was transferred to a 1.5 mL centrifuge tube, and the tube with lysate was centrifuged at 13,000 rpm for 10 min, and the supernatant was collected. The supernatant was taken for western blot detection of the phosphorylation of STAT3. The results were shown in Figure 14. The results show that KP4 cell line is most sensitive to the stimulation of human LIF protein.
Example 12 Recombinant Expression and Verification of the 38E10E1C11 mAb antibody and the P36-033 mAb The light chain gene and the heavy chain gene of the 38E10E1C11 mAB and the P36-033 mAb were constructed into the eukaryotic expression vector PCDNA3.1+ by identical recombination technique. The recombinant antibodies were expressed by Thermo's ExpiCHO expression system, and the recombinant antibodies were purified by Protein G affinity tography. Endotoxin removal of the purified antibodies is carried out using Endotoxin Removal Beads ed by Smart-lifesciences company.
The specific experimental method referred to Example 10.2. Figure 15 shows that the 38E10E1C11 antibody (labeled as 38E10E1C11R) recombinantly expressed by CHO cells was e of inhibiting phosphorylation of STAT3 in KP4 cells induced by human LIF protein.
Example 13 Identification of epitope recognized by 38E10E1C11 antibody It was med by the preliminary experiments that the 38E10E1C11 dy (SEQ ID NOs: 41 and 43) recognized the linear epitope on the surface of human LIF protein (SEQ ID NO: 58). The dy could not recognize mouse LIF protein, and could block the binding of human LIF protein and human LIFR protein. According to these three points, combined with the analysis of a y of online protein linear epitope prediction software, it was ated that the ition epitope of the antibody was located in the 160-202amino acid sequence of LIF protein, so the invention synthesized the following heterozygous LIF protein. Mut3(SEQ ID NO: 59) is to replace the182-202 amino acid sequence of human LIF protein with that of mouse LIF protein, mut4 (SEQ ID NO: 60) is to replace the166-202 amino acid sequence of human LIF protein with that of mouse LIF protein. The plasmids containing mut3, mut4 and full-length human LIF protein were transfected into 293T cells. After three days of culture, the culture supernatant of 293T cells was taken for GE electrophoresis and western blot. The culture supernatant of 293T cells was used as negative control, 38E10E1C11 as primary antibody and HRP labeled Goat anti-mouse Fab as secondary antibody. At the same time, the M1 cell eration experiment was used to detect the activity of the hybrid protein and verify the neutralizing activity of 38E10E1C11 to the hybrid protein. Set up multiple groups of control wells at the same time, control wells adding human recombinant LIF protein (rhLIF, purchased from Yiqiao Shenzhou, the product number is: 14890-HNAH), control wells adding rhLIF and 38E10E1C11, and control wells t adding rhLIF and LIF antibody. The results showed that the 38E10E1C11 antibody could recognize the denatured full-length LIF protein and mut3 protein, but could not recognize mut4 n (Subfigure A in Figure 16). The M1 cell proliferation ment shows that 38E10E1C11 antibody can reverse the inhibition of the full-length LIF protein and Mut3 protein on the proliferation of M1 cells but cannot reverse the inhibitory effect of the Mut4 protein gure B in Figure 16). In summary, the recognition epitope of 38E10E1C11 antibody was located in the167-181 amino acid sequence of human LIF protein, that is, the amino acids TYGPDTSGKDVFQKK (SEQ ID NO:61). e 14 Design and expression purification of humanized LIF antibody Monoclonal antibody 38E10E1C11 obtained from mouse immunization was humanized. Humanization was performed by standard CDR grafting method. The heavy and light chain s were cloned from 38E10E1C11 hybridoma by standard molecular cloning ques and sequenced by Sanger method. BLAST searches were then med on the human heavy and light chain variable sequences and three or four sequences were selected as the receptor frames for humanization. The heavy and light chain CDR1, CDR2 and CDR3 of 38E10E1C11 were cloned into three different heavy chain receptor frames (H1-H3) and four different light chain frames (L1-L4), while the HCDR2 of 38E10E1C11 (amino acid sequence before mutation as shown in SEQ ID NO:45) was point mutated (the mutated amino acid sequence as shown in SEQ ID NO:5), the human IgG1 isoform was ed for the heavy chain constant region and the human kappa chain was selected for the light chain constant . 293S cells were co-transfected with expression vectors containing the gene of the humanized antibody heavy chain and the humanized antibody light chain. The gene sequences of the heavy and light chain variable region, the amino acid sequence of the variable , the full-length gene sequence and the full-length amino acid sequence were shown in Table 1. The expression levels, antigen g ability and thermal stability of the twelve different antibodies combinations in 293S cells were then examined. The 38E10E1C11 chimeric antibody (Chimeric) was used as a positive control, and all 38E10E1C11 chimeric antibodies were abbreviated as 38E chimeric antibody or 38E Chimeric (SEQ ID NO: 52 and SEQ ID NO:50) in subsequent assays. The medium was collected and the expression levels of IgG n were quantified on a Gator (similar to Octet) and corrected by ELISA. The antigen binding ability of the different combinations was compared by ELISA (Table 2, Table 3) Enzyme linked immunosorbent assay (ELISA): Each well was coated with 100 μL of 0.5 μg/ml antigen and incubate overnight at 4°C, and the plates were washed three times with 300 μL of Wash . The plates were closed with 200 μL of Closure Buffer (2% bovine serum n) for 60 min at room temperature. The plates were washed three times with 300 μL of Wash Buffer. 100 μL of diluted anti-LIF antibody at different concentrations was added to each well and incubate for 1 hour at room temperature. The plates were washed 4 times with 300 μL of Wash Buffer. 100 μL of HRP-labeled goat anti-human Fc secondary antibody at a on of 1:5000 was added and incubate for 1 hour at room ature. The plates were washed 6 times with 300 μL of Wash Buffer. 100 μL of H2O2-Amplx Color Development Solution was added for developing the color for 10 min at room temperature under dark conditions. The OD 450 value was read by an enzyme marker. Heat treatment: the expression medium was heated on the PCR machine at 70°C for 5 min and then rapidly cooled to room temperature. m subsequent ELISA assays as above.
Table 1. Expression levels of different combinations of humanized light and heavy chains in 293S cells Gene antibody Amino acid Full length sequence of Full length concentrati sequence of amino acid variable gene sequence on in No. variable region sequence of region of of humanized culture of humanized humanized zed antibody supernatan antibody antibody dy t (μg/ml) SEQ ID SEQ ID NO:26 SEQ ID NO: 25 heavy SEQ ID NO: 23 NO:24 (full heavy (full heavy chain (VH1,aa) (VH1,nt) chain 1,nt) chain 1,aa) H1L1 95.5 SEQ ID SEQ ID NO:10 SEQ ID NO:9 light SEQ ID NO: 7 NO:8 (full light chain (full light chain chain (VL1,aa) (VL1,nt) 1,nt) 1,aa) SEQ ID SEE ID NO:30 SEQ ID NO: 29 heavy SEQ ID NO: 27 NO:28 (full heavy (full heavy chain a) (VH2,nt) chain 2,nt) chain 2,aa) H2L1 120 SEQ ID SEQ ID NO:10 SEQ ID NO: 9 light SEQ ID NO: 7 NO:8 (full light chain (full light chain chain (VL1,aa) (VL1,nt) 1,nt) 1,aa) SEQ ID SEQ ID NO:34 SEQ ID NO: 33 heavy SEQ ID NO: 31 NO:32 (full heavy (full heavy chain (VH3,aa) (VH3,nt) chain 3,nt) chain 3,aa) H3L1 108 SEQ ID SEQ ID NO:10 SEQ ID NO: 9 light SEQ ID NO: 7 NO:8 (full light chain (full light chain chain (VL1,aa) (VL1,nt) 1,nt) 1,aa) SEQ ID SEQ ID NO:26 SEQ ID NO: 25 heavy SEQ ID NO: 23 NO:24 (full heavy (full heavy chain (VH1,aa) (VH1,nt) chain 1,nt) chain 1,aa) H1L2 110 SEQ ID SEQ ID NO:14 SEQ ID NO: 13 light SEQ ID NO: 11 NO:12 (full light chain (full light chain chain (VL2,aa) (VL2,nt) 2,nt) 2,aa) SEQ ID SEQ ID NO:30 SEQ ID NO: 29 heavy SEQ ID NO: 27 NO:28 (full heavy (full heavy chain (VH2,aa) (VH2,nt) chain 2,nt) chain 2,aa) H2L2 89.4 SEQ ID SEQ ID NO:14 SEQ ID NO:13 light SEQ ID NO: 11 NO:12 (full light chain (full light chain chain (VL2,aa) (VL2,nt) 2,nt) 2,aa) SEQ ID SEQ ID NO:34 SEQIDNO: 33 heavy SEQ ID NO: 31 NO:32 (full heavy (full heavy chain (VH3,aa) (VH3,nt) chain 3,nt) chain 3,aa) H3L2 95.3 SEQ ID SEQ ID NO:14 SEQ ID NO: 13 light SEQ ID NO: 11 NO:12 (full light chain (full light chain chain (VL2,aa) t) 2,nt) 2,aa) SEQ ID SEQ ID NO:26 SEQ ID NO: 25 heavy SEQ ID NO: 23 NO:24 (full heavy (full heavy chain (VH1,aa) (VH1,nt) chain 1,nt) chain 1,aa) H1L3 115 SEQ ID SEQ ID NO:18 SEQ ID NO: 17 light SEQ ID NO: 15 NO:16 (full light chain (full light chain chain (VL3,aa) (VL3,nt) 3,nt) 3,aa) SEQ ID SEQ ID NO:30 SEQ ID NO: 29 heavy SEQ ID NO: 27 NO:28 (full heavy (full heavy chain a) (VH2,nt) chain 2,nt) chain 2,aa) H2L3 123 SEQ ID SEQ ID NO:18 SEQ ID NO: 17 light SEQ ID NO: 15 NO:16 (full light chain (full light chain chain (VL3,aa) (VL3,nt) 3,nt) 3,aa) SEQ ID SEQ ID NO:34 SEQ ID NO: 33 heavy SEQ ID NO: 31 NO:32 (full heavy (full heavy chain (VH3,aa) (VH3,nt) chain 3,nt) chain 3,aa) H3L3 97.6 SEQ ID SEQ ID NO:18 SEQ ID NO: 17 light SEQ ID NO: 15 NO:16 (full light chain (full light chain chain (VL3,aa) (VL3,nt) 3,nt) 3,aa) SEQ ID SEQ ID NO:26 SEQ ID NO: 25 heavy SEQ ID NO: 23 NO:24 (full heavy (full heavy chain (VH1,aa) (VH1,nt) chain 1,nt) chain 1,aa) H1L4 183 SEQ ID SEQ ID NO:22 SEQ ID NO: 21 light SEQ ID NO: 19 NO:20 (full light chain (full light chain chain a) (VL4,nt) 4,nt) 4,aa) SEQ ID SEQ ID NO:30 SEQ ID NO: 29 heavy SEQ ID NO: 27 NO:28 (full heavy (full heavy chain (VH2,aa) (VH2,nt) chain 2,nt) chain 2,aa) H2L4 `155 SEQ ID SEQ ID NO:22 SEQ ID NO: 21 light SEQ ID NO: 19 NO:20 (full light chain (full light chain chain (VL4,aa) (VL4,nt) 4,nt) 4,aa) SEQ ID SEQ ID NO:34 SEQ ID NO: 33 heavy SEQ ID NO: 31 NO:32 (full heavy (full heavy chain (VH3,aa) (VH3,nt) chain 3,nt) chain 3,aa) H3L4 172 SEQ ID SEQ ID NO:22 SEQ ID NO: 21 light SEQ ID NO: 19 NO:20 (full light chain (full light chain chain a) (VL4,nt) 4,nt) 4,aa) SEQ ID SEQ ID NO:53 SEQ ID NO: 52 NO:49 SEQ ID NO: 48 heavy (38E (38E Chimeric (38E (38E Chimeric chain Chimericfull full heavy Chimeric VH, aa) 38E heavy chain,nt) chain,aa) VH,nt) Chim 97.4 SEQ ID eric SEQ ID NO:51 SEQ ID NO: 50 NO:47 SEQ ID NO: 46 light (38E (38E Chimeric (38E (38E Chimeric chain Chimericfull full light Chimeric VL, aa) light chain,nt) chain,aa) VL,nt) Table 2. ELISA results of ent combinations of humanized light and heavy chains via 293S cell expression supernatant (non-heated) Antibody concentration （μg/mL） 1 0.2 0.04 0 Clone No./ non-heated H1L1 11840 10574 6849 2134 H2L1 11953 10737 6369 H3L1 11367 10041 6222 H1L2 12118 11246 6682 H2L2 11699 10321 6517 H3L2 11266 10575 6599 H1L3 11846 10790 6435 H2L3 11967 11286 6266 H3L3 11280 10978 6755 H1L4 11929 10931 7321 H2L4 11821 10859 6877 H3L4 11918 11226 7055 38E Chimeric 11353 10414 6783 Table 3. ELISA results of different combinations of humanized light and heavy chains via 293S cell expression atant (heated) Antibody concentration （μg/mL） 1 0.2 0.04 0 Clone No/heated H1L1 9796 9007 5970 27.7 H2L1 9647 8560 5442 H3L1 9131 7903 5192 H1L2 9833 8925 5493 H2L2 9779 8716 5259 H3L2 9448 8775 5065 H1L3 9627 8748 4978 H2L3 9731 9153 4866 H3L3 9543 8898 5393 H1L4 10526 9705 5670 H2L4 10572 9234 5702 H3L4 9948 9061 5299 38E Chimeric 10567 8845 4727 Example 15 terization of the selected zed candidate antibodies with purified IgG samples Based on the binding affinity, tage of humanization, antibody expression level and thermal stability data, the following five candidate dies were selected for the next characterization step: H1L1, H1L4, H2L4, H3L2, H3L4, and the five candidate antibodies were renumbered as 38E HuH1L1 (SEQ ID NOs: 25 and 9), 38E HuH1L4 ( SEQ ID NOs: 25 and 21), 38E HuH2L4 (SEQ ID NOs: 29 and 21), 38E HuH3L2 (SEQ ID NOs: 33 and 13), and 38E HuH3L4 (SEQ ID NOs: 33 and 21).
Then the selected VH/VL plasmids were co-transfected with 293S cells, the cell culture supernatant was harvested, and the antibody was purified by protein A affinity chromatography. The purified antibody was used for binding ELISA analysis to compare the specific binding ability of the humanized antibodies to the 38E chimeric antibody. The invention also underwent some preliminary analyses to compare their thermal stability and non-specific binding. The results showed that the candidate antibodies being purified and the 38E chimeric antibody had very similar antigen-binding properties (Figure 17A, 17C). After treatment at 70°C for 5 min, all five humanized antibodies showed similar binding ability with the ic dies (Figure 17B, 17D).
Example 16 Evaluation of non-specific binding of humanized candidate antibodies LIF-negative HEK293 cells FACS was used for preliminary assay to assess the risk of potential non-specific binding of the antibody.
HEK293 cells were digested with trypsin, washed twice with PBS containing 1% FBS, resuspended, adjusted to a cell density of 1.5-2x106 cells/mL, and added to a 96-well U-shaped plate. The concentration of antibody to be detected was adjusted to μg/mL, and then 3-times gradient dilution was performed for a total of 8 concentrations, and a blank control and a negative control (Rituxan) were set up. The diluted antibody and blank l were added to the cells in 96-well plates, and 100 μL of antibody was added to each well. The cells were incubated at 4 °C for 1 hour, centrifuged at 1000rpm for 5 s, the supernatant was carefully ded and washed twice with PBS ning 1% FBS, and finally resuspended with 200 μL of PBS containing 1% FBS, finally, resuspend the cells with 200μL of PBS containing 1% FBS, and flow cytometric analysis was performed. In the ecific binding FACS assay of HEK293 cell, 38E , 38E HuH3L2, 38E HuH3L4, 38E HuH1L4, 38E chimeric antibody and negative control (Rituxan) had r ecific binding affinity for HEK293 cells, while 38E HuH2L4 had a higher non-specific binding affinity for HEK293 cells (Subfigure A and B in Figure 18).
Example 17 CE-SDS analysis of antibody purity The working concentration of CE-SDS analysis was 1 mg/mL, the dy samples were diluted to the specified tration with the loading buffer.
Preparation of non-reduced CE-SDS ophoresis samples: 95μL of diluted sample solution was taken, 5μL of 0.8M ammonium iodoacetate aqueous solution and 5μL of internal reference were added, ed and mixed well. 95μL of blank control was taken, 5μL of 0.8M ammonium iodoacetate aqueous solution and 5μL of internal nce were added, vortexed and mixed well for non-reduced blank control. Then heated in metal bath at 70 °C for 5 minutes, cooled to room temperature, and centrifuged at 6000 rpm for 1 minute.
Preparation of the reduced sample solution: 95 μL of diluted sample solution was taken, 5 μL of 2-mercaptoethanol solution and 5 μL of internal reference were added, vortexed and mixed well. 95μL of blank control was taken, and 5μL of 2-mercaptoethanol solution and 5μL of internal reference were added, ed and mixed well for reduced blank control. Then heated in metal bath at 70 °C for 15 minutes, cooled to room ature, and centrifuged at 6000rpm for 1 .
Sample analysis: 75 μL of sample was added to the test tube, and the test tube was placed into the test cup. The test cup was carefully inserted into the injection tray, and the test program was run with a reduced sample injection duration of 30 seconds and a non-reduced sample injection duration of 40 seconds, capillary temperature of °C, sample temperature of 20°C, focusing voltage of 15 KV, focusing time of 40 minutes, and data were collected with a PDA detector at 214 nm. CE s are shown in Table 4, Table 5.
Table 4. Results of the reduced CE-SDS Peak# Size(KDa) Peak Area (%) Peak ID 1 27.77 35.5 LC 38E chimeric 2 62.43 64.5 HC 1 25.6 0.57 LMC 2 27.85 32.02 LC 38E HuH1L1 3 36.03 1.41 LMC 4 61.41 66 HC 1 27.41 31.7 LC 38E HuH3L2 2 33.84 0.35 LMC 3 62.49 67.95 HC 1 27 34.93 LC 38E HuH3L4 2 62.06 65.07 HC Table 5. Results of the non-reduced CE-SDS 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 Example 18 Thermal stability analysis by differential ng fluorescence (DSF)/static light scattering (SLS) que Samples were submitted to UNcle Systems (Unchained Labs) for analysis. The temperature range of 25°C to 95°C was monitored for DSF and SLS at 1°C/min.
UNcle ed SLS at 266 nm and 473 nm. Tm and Tagg were calculated and analyzed using UNcle analysis software.
IgG is with multiple structural domains, each has its own melting ature (Tm.) The CH2 structural 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 larger range of Tm of about 50-85 °C due to their more variable sequences. Therefore, the Tm values measured by various analytical techniques are usually the "apparent" transition temperatures, rather than the true Tm values of each structural domain. It is clear that even this DSF analysis can produce more than one Tm value, only Tm1 is used to evaluate the thermal stability of therapeutic antibodies. Tagg is the temperature at which SLS starts to detect aggregation. 6 measures SLS at 266nm, which is more sensitive and more suitable for ing smaller aggregated particles. Tagg473 measures SLS at 473nm, which is more le for detecting larger les.
As shown in Table 6, all three humanized candidate antibodies have higher melting ature (Tm1) and less aggregation risk than the 38E chimeric antibody.
Table 6: Results of Differential Scanning Fluorescence (DSF)/Static Light Scattering (SLS) analysis DSF( °C) SLS( °C) Sample Tm1 Tm2 Tm3 Tagg 266 Tagg 473 38E chimeric 69.3 68.5 69.8 38E HuH1L1 72.7 82.8 72.5 73.6 38E HuH3L2 72 82.4 73.3 73.7 38E HuH3L4 71.5 82.1 69.8 70.9 Example 19 Analysis of aggregation tendency of antibodies using dynamic light scattering technique (DLS) DLS was performed on the UNcle system (Unchained Labs). DLS was measured at 25°C. Data were calculated and analyzed using UNcle analysis software. Dynamic Light Scattering (DLS) is used to detect the aggregation in antibody samples. The "mode diameter" refers to the er of the protein particle, and the "mass percentage" refers to the percentage of each le size fraction. The "PDI" refers to the polydispersity index, the higher the index, the more polydispersed the sample is. If the PDI is not greater than 0.25, the sample can be considered as mono-disperse. As shown in Table 7, all four antibody samples had a main "peak" (mass on over 99%), with 38E HuH3L4 having a better PDI than the chimeric dy, 38E HuH3L2 being similar to the chimeric antibody, and 38E HuH1L1 having a worse PDI than the chimeric antibody.
Table 7: Results of Dynamic light scattering technique (DLS) is Peak1 Peak2 mode diameter mass PDI mode diameter mass Sample (nm) percentage (nm) percentage (%) (%) 38E chimeric 10.41 99.90 0.229 38E HuH1L1 9.68 99.15 0.383 101.16 0.96 38E HuH3L2 10.41 99.62 0.28 99.18 0.38 38E HuH3L4 10.41 100.00 0.043 Example 20 dy Affinity Assay The affinity of anti-LIF antibody to human LIF protein was determined using Gator. The anti-human LIF antibody was first diluted to 5ug/mL with PBS and then added to A-F wells in second column of the 96-well plate (200μL per well). The human LIF protein trations were gradient diluted with PBS to 100, 50, 25, 12.5 and 6.25μg/mL, respectively, and the diluted LIF protein was added to wells A-E wells in the fourth column of the 96-well plate (100μL per well) and PBS was added to F well as blank control. PBS was added to A-F wells in the first and third columns (200μL per well). The 96-well plates were placed into the instrument and detected with anti-human Fc biosensor. The results were shown in Table 8, which showed that the affinity of the three humanized antibodies was close to that of the chimeric antibodies.
Table 8: Humanized anti-LIF antibody affinity assay 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 Example 21 Expression purification of humanized antibodies and 38E10E1C11 antibodies The variable regions of the light and heavy chains of the zed antibodies 38E HuH3L2 and 38E HuH3L4 were linked with the constant regions of the mouse antibodies (the constant region of the heavy chain was mouse IgG1, the constant region of the light chain was kappa chain) and cloned into the PCDNA.3.4 vector, respectively, named 38E HuH3L2-m (the full-length gene sequences encoding the heavy and light chains of the 38E HuH3L2-m antibodies 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 -m (the full-length gene sequences of the heavy and light chains 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 antibodies are shown in SEQ ID NO:35 and SEQ ID NO:39, tively). Gene transfection and antibody expression were performed using the Expi CHO Expression Kit from thermo fisher.
Cell e atant was collected and the dy was purified using a protein G ty chromatography column. The purified antibody was concentrated and exchanged by ultrafiltration using Amicon® Ultra iltration tubes, and the antibody was finally dissolved in PBS of pH 7.4. The 38E10E1C11 antibody was also expressed and purified in the same manner.
Example 22 Humanized anti-LIF antibody es with LIFR for binding to human LIF protein The recombinant human LIF protein is coated at a tration of 1 μg/mL in enzyme labeled plates, and 50 μL/well of recombinant human LIFR protein at a concentration of 0.6125 μg/mL (fusion expressed with human Fc, purchased from ACRO, item number: LIR-H4252) is added, meanwhile 100 μL/well of different LIF antibodies 38E HuH3L2-m (SEQ ID NOs: 35 and 37), 38E -m (SEQ ID NOs: and 39), 38E10E1C11 (SEQ ID NOs: 41 and 43), P36-033 (SEQ ID NOs: 54 and 56) at different concentrations were added. Anti-CD3 antibody was used as negative control (purchased from BioLegend, No. ). The plates were incubated for 2 h at room temperature and washed four times with PBST, HRP-labeled goat anti-human Fc antibodies were added, and the plates were incubated for 1 h at room temperature and washed four times with PBST. TMB colored solution was added and color developing for 10 min at room ature. The absorption value at 450 nm was read by an enzyme . The data were analyzed and plotted using Origin pro 9 software. The results were detailed in Figure 19. The results showed that 38E10E1C11, 38E HuH3L2-m, 38E HuH3L4-m were able to inhibit the binding of recombinant human LIF to human LIFR with IC50 of 0.074 μg/ml, 0.145 μg/ml and 0.103 μg/ml, respectively. P36-033 had a weak inhibitory effect and the negative control D3 antibody could not inhibit the binding of recombinant human LIF to human LIFR.
Example 23 Humanized anti-LIF dy does not compete with GP130 to bind human LIF protein The recombinant human LIF protein was coated at a concentration of 1 μg/mL in the enzyme labeled plate, and 50 l of recombinant human GP130 n at a concentration of 12 μg/mL (fusion expressed with human Fc, purchased from Yijiao Shenzhou, item number: H03H) was added , meanwhile 100 l of LIF antibodies 38E HuH3L2-m ((SEQ ID NOs: 35 and 37), 38E HuH3L4-m (SEQ ID NOs: 35 and 39) and P36-033 (SEQ ID NOs: 56 and 54) at different concentrations were added, anti-CD28 antibody was as a negative control (purchased from BioLegend, item no. 302914). The plates were incubated for 2 h at room temperature and washed with PBST four times. HRP-labeled goat anti-human Fc antibodies were added and incubated for 1 h at room temperature, and washed four times with PBST.
TMB colored solution was added and color developing for 10 min at room temperature. The absorption value at 450 nm was read by an enzyme marker. The data were analyzed and plotted using Origin pro 9 software. The results were detailed in Figure 20. The results showed that the humanized antibodies 38E HuH3L2-m, 38E HuH3L4-m and the negative control antibody of anti-CD28 dy did not inhibit the binding of recombinant human LIF to human GP130 protein, and P36-033 could t the binding of recombinant human LIF to human GP130 protein.
Example 24 Antigen recognition specificity assay of humanized anti-LIF antibody Human LIF, human IL-6, human OSM and human CNTF (all four proteins were sed from Yijiao Shenzhou, item numbers 14890-HNAH; 10395-HNAE; 10452-HNAH; 11841-H07E, respectively) were coated at a concentration of 1 μg/mL in enzyme labeled plates, and different concentrations of LIF antibodies 38E10E1C11, 38E huH3L2-m, 38E huH3L4-m were incubated at room temperature for 1 h. After washed four times with PBST, HRP-labeled goat anti-mouse Fab secondary dies were added and incubated at room temperature for 1 h. After washed four times with PBST, TMB colored solution was added and color developing for 10 min at room temperature. The absorption value at 450 nm was read by the enzyme maker.
The data were analyzed and plotted using Origin pro 9 software. The results were shown in Figure 21, the result shows that 38E10E1C11, 38E huH3L2-m and 38E huH3L4-m antibodies only bind to human LIF protein but not to human IL-6, OSM and CNTF.
Example 25 Identification of es Recognized by Humanized anti-LIF Antibody In the us experiments, the invention found that the 38E10E1C11 antibody recognizes a linear epitope of LIF protein, so r the 38E humanized antibody still recognizes the linear epitope of LIF protein y needed to be verified. 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 ve control of 293T cell culture supernatant were taken, 5xSDS-PAGE loading buffer was added, and boiled for 10 minutes. Then 10 μL of sample was taken for GE electrophoresis, and then the electrophoretic bands were transferred to PVDF membrane for western blot detection. The primary antibody for ion was 38E huH3L2 or 38E huH3L4 antibody at a concentration of 1 μg/mL, and was incubated for 2 hours at room temperature. Then washed three times in PBST buffer and diluted HRP-labeled sheep uman Fc secondary antibody at a dilution ration of 1:3000 was added, incubated with the secondary antibody for 2 hours at room temperature, washed three times in PBST buffer and ed Chemiluminescent solution (Perice, item no. 34079) was added and incubated. An Amersham Imager 600 ultra-sensitive multifunctional imager was used for detection and photograph. The results were shown in ure A in Figure 22, the result shows that both humanized antibodies could recognize the denatured human LIF protein and Mut3 protein but not Mut4 protein. The same results were obtained in the M1 cell proliferation assay, and the results were detailed in subfigure B in Figure 22. ore, the epitope sequences recognized by the 38E huH3L2 and 38E huH3L4 antibodies were determined to be TYGPDTSGKDVFQKK (SEQ ID NO: 61).
Example 26 STAT3 activation inhibition assay of KP4 cells to detect humanized anti-LIF antibody activity After digestion and centrifugation of KP4 cells, cells were pended and plated in 12-well plates at 1 mL, 5x105cells/well. The plates were incubated at 37°C, % CO2 overnight. The next day, the original medium was discarded, cell medium containing 50ng/mL recombinant human LIF protein and different concentrations of anti-LIF humanized antibodies was added respectively. Control wells t recombinant human LIF protein and with only inant human LIF n and without antibodies were set up, and the plates were incubated for 30min at 37 °C incubator. Then the medium was removed, 100μL of 1x cell lysis solution was added to each well, and the mixture was lysed for 30min on ice. The lysate was transferred to a 1.5 mL centrifuge tube and centrifuged at 13,000 rpm for 10 min, and the supernatant was collected. The supernatant was taken for western blot detection to detect the phosphorylation of STAT3. The s showed that the zed dies 38E huH3L4 and 38E huH3L2 were able to inhibit LIF protein-induced phosphorylation of STAT3 (Figure 23).
Example 27 M1 cell proliferation assay to detect the activity of humanized anti-LIF antibody After centrifugation of M1, washed with RPMI1640 medium twice, and 96-well plates were inoculated at a density of 2.5x105 cells/mL. 80 μL of the cells were inoculated in each well, and medium containing 4 ng/mL of recombinant human LIF protein and different concentrations of anti-LIF antibodies were added to make the final volume of each well to 160 μL. While control wells without LIF were set up and incubated at 37 °C for 72 hours and proliferation was ed by adding CCK-8. The results were detailed in Figure 24, the result shows that both humanized antibodies 38E -m and 38E huH3L2-m were able to reverse the proliferation inhibition of M1 cells by human LIF protein with EC50 of 6.52 μg/mL and 8.93 μg/mL, tively.
Example 28: Inhibition effect of duced STAT3 phosphorylation by LIF antibody 100,000 KP4 cells were inoculated in 96-well plates; and gradient diluted LIF antibody was incubated with 20-100 ng/ml of LIF protein at room temperature for 0.5-1h to form LIF-Ab e. LIF-Ab mixture was added to cell wells and stimulated for 5-30 min at 37°C. Detecting P-STAT3 and Total-STAT3 protein expression levels ing to P-STAT3(TYR705) KITS( Cisbio) and Total-STAT3 KITS (Cisbio) instructions. The emission signal ratio of the donor and acceptor for each well was calculated: Ratio= Signal 665nm/Signal 620nm *104. Prism software was used to generate data graphs and count the inhibition rate of LIF antibody. The results showed that the LIF antibody 38E HuH3L4 has inhibition effect of phosphorylation of STAT3 phosphorylation induced by LIF, as shown in Figure 25, with an IC50 of 3.415 nM. e 29: Detection of ADCC Activity of LIF Antibody LIF binds to GP130 and LIFR, while humanized LIF antibody blocks LIF binding to LIFR, but not LIF binding to GP130. Detecting r humanized LIF antibody binds to the cell surface mediated by LIF and thus ADCC works. Antibodies Erbitux (Epiduo®, Merck Serono, positive l) and Human IgG2 (Cat#HG2K, Sino, negative control) were sequentially diluted with ADCC buffer (RPMI-1640+1% FBS); and the 38E huH3L4 antibody was then diluted in triplicate and eight gradients with ADCC buffer containing LIF protein, and set aside. DLD-1 cells were digested with trypsin (Cat#25200072, GIBCO), and after the on was terminated, the cells were blown apart and collected into a centrifuge tube, centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the cells were re-suspended with ADCC buffer and counted. The cell concentration was ed and set aside. PBMC cells (Cat#SLB-HP010B, Shanghai SAILYBIO Ltd.) were resuscitated, and 10mL of ADCC buffer was added, centrifuged at 2000rpm for 10min, and the supernatant was discarded. The PBMC cells were pend in ADCC buffer and counted. The cell concentration was adjusted and set aside; and take out the 96-well U-bottom cell culture plate, 50μl of target cells DLD-1, 50μl of antibody, and 50μl of PBMC effector cells were added in turn. The ratio of PBMC effector cells and target cells was 30:1.The reaction was carried out in a 5% CO2 incubator at 37 °C for 6h. LDH was detected by Cyto Tox96 dioactive Cytotoxicity Assay Kit (Cat#G1780, Promega) and the absorbance values were measured at 490nm using an enzyme marker.
The mean absorbance values of each parallel well were calculated so that the average absorbance values of all experimental wells, target cell LDH spontaneous release wells (TCR), and effector cell LDH spontaneous release wells (ECR) were subtracted from the average absorbance values of blank medium (CMB). The e absorbance value of the target cell LDH maximum release wells (TCM) was subtracted from the e absorbance value of the volume ted control wells (VCC). Cytotoxicity (%) from each concentration of antibody was ated using the above corrected values according to the following formula.
Cytotoxicity (%) = (A - B - C)/(D - C) × 100% A: average absorbance value after correction of experimental wells B: average ance value of the corrected effector cell LDH spontaneous release wells C: average absorbance value of the corrected target cell LDH spontaneous release wells D: average absorbance value of the corrected LDH l release pore of the target cell As shown in Figure 26, 38E huH3L4 antibody had no ADCC activity.
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Stimulation of the JAK/STAT pathway by LIF and OSM in the human granulosa cell line . J Reprod Immunol. 2015; 108:48-55. 3. Liu SC, Tsang NM, Chiang WC, Chang KP, Hsueh C, Liang Y, Juang JL, Chow KP, Chang YS. Leukemia inhibitory factor promotes nasopharyngeal carcinoma progression and esistance. J Clin Invest. 2013; 123(12):5269-83. 4.Shi Y, Gao W, Lytle NK, Huang P, Yuan X, Dann AM, et al. Targeting LIF-mediated ine interaction for pancreatic cancer therapy and monitoring. Nature. 2019; 569(7754):131-135.
. Cartwright an C, Sheppard A, RivettD, Jones K,Dalton S. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 2005; 132:885–96. 6. Kuphal S, Wallner S, Bosserhoff AK. Impact of LIF (leukemia inhibitory factor) sion in malignant ma. Exp Mol Pathol 2013; 95:156–65. 7. Liu B, Lu Y, Li J, Liu Y, Liu J,WangW. Leukemia inhibitory factor promotes tumor growth and asis inhuman osteosarcoma via activating STAT3. APMIS 2015;123:837– 8. Morton SD, Cadamuro M, Brivio S, Vismara M, Stecca T, Massani M, et al.Leukemia inhibitory factor protects cholangiocarcinoma cells from drug d apoptosis via a PI3K/AKT-dependent Mcl-1 activation. Oncotarget. :26052–64. 9. Kamohara H, Ogawa M, Ishiko T, Sakamoto K, Baba H. Leukemia inhibitory factor functions as a growth factor in pancreas carcinoma cells: involvement of regulation of LIF and its receptor expression. Int J Oncol. 2007;30:977–83.
. Shin JE, Park SH, Jang YK. Epigenetic up-regulation of leukemia inhibitory factor (LIF) gene during the progression to breast cancer. Mol Cells 2011; 31:181–9. 11.Li X, Yang Q, Yu H, Wu L, Zhao Y, Zhang C, Yue X, Liu Z, Wu H, Haffty BG, Feng Z, Hu W. LIF promotes genesis and metastasis of breast cancer through the AKT-mTOR pathway. Oncotarget. 2014; 5(3):788-801. 12. Liu SC, Hsu T, Chang YS, Chung AK, Jiang SS, OuYang CN, Yuh CH, Hsueh C, Liu YP, Tsang NM. Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling. Nat Commun. 2018; 9(1):5105. 13. Viswanadhapalli S, Luo Y, Sareddy GR, Santhamma B, Zhou M, et al. EC359: A First-in-Class Molecule Inhibitor for Targeting Oncogenic LIFR Signaling in Triple-Negative Breast Cancer. Mol Cancer Ther. 2019; 18(8):1341-1354.

Claims (33)

What is claimed is:
1. An isolated antibody or antigen-binding fragment thereof that binds at an epitope represented by an amino acid ce TYGPDTSGKDVFQKK (SEQ ID NO: 61) of human LIF protein or at an epitope of the corresponding amino acid sequence of a different ian species.
2. An isolated antibody or antigen-binding fragment thereof, comprising: 1) (a) LCDR1 comprising SEQ ID NO: 1, (b) LCDR2 sing 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; 2) (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; or 3) (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.
3. The isolated antibody or antigen-binding fragment thereof of claim 2, comprising: 1) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 23; 2) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 7, and a heavy chain le region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 27; 3) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; 4) a light chain le region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23; 5) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 27; 6) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region (VH) that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; 7) a light chain variable region (VL) that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 23; 8) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 27; 9) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; 10) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 23; 11) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 27; 12) a light chain variable region (VL) that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 31; 13) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 46, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 48; 14) a light chain variable region (VL) that ses an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 74, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 75; or 15) a light chain variable region (VL) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 82, and a heavy chain variable region (VH) that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO:83.
4. The isolated dy or antigen-binding fragment thereof of any one of claims 2 to 3, comprising: 1) a light chain that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 2) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 3) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 9, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 4) a light chain that comprises an amino acid ce with at least 85% identity to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 5) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 6) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 13, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 7) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 8) a light chain that ses an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 9) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 17, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 10) a light chain that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 21, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 25; 11) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 21, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 29; 12) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 21, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 33; 13) a light chain that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 37, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 35; 14) a light chain that ses an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 39, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid ce of SEQ ID NO: 35; 15) a light chain that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 50, and a heavy chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 52; or 16) a light chain that comprises an amino acid sequence with at least 85% identity to an amino acid sequence of SEQ ID NO: 54, and a heavy chain that comprises an amino acid sequence with at least 85% ty to an amino acid sequence of SEQ ID NO: 56.
5. The isolated antibody or antigen-binding fragment thereof of claim 2 comprising a) a light chain variable region (VL) represented by SEQ ID NO: 7, and a heavy chain variable region (VH) represented by SEQ ID NO: 23; or b) a light chain variable region (VL) represented by SEQ ID NO: 11, and a heavy chain variable region (VH) represented by SEQ ID NO: 31; or c) a light chain variable region (VL) represented by SEQ ID NO: 19, and a heavy chain variable region (VH) represented by SEQ ID NO: 31.
6. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the isolated antibody is an IgG.
7. The ed antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the isolated dy is an IgG1, IgG2 or IgG4.
8. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the isolated antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human ered antibody, a human antibody, a bispecific antibody, Fv, a single chain antibody (scFv), a Fab, a Fab’, a Fab’-SH or a F(ab’)2.
9. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 8, which is leukemia inhibitory factor (LIF) antagonist.
10. The isolated dy or antigen-binding fragment thereof of any one of claims 1 to 8, which is e of inhibiting the expression of LIF and/or blocking the ty of LIF.
11. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 8, which is capable of competing or cross competing for binding to LIF.
12. A tide composition comprising a nucleotide molecule encoding the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 11.
13. The nucleotide composition of claim 12, comprising: 1) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 2) a first nucleic acid molecule sing DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; 3) a first nucleic acid le comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31; 4) a first nucleic acid le comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second c acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 5) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second c acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; 6) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31; 7) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid ce of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 8) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid ce of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid ce of SEQ ID NO: 27; 9) a first nucleic acid molecule comprising DNA ng a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 15 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid ce of SEQ ID NO: 31; 10) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid le comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23; 11) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27; 12) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 and a second nucleic acid molecule sing DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31; 13) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 46 and a second nucleic acid molecule comprising DNA encoding a heavy chain le region (VH) as represented by an amino acid sequence of SEQ ID NO: 48; 14) a first c acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 74 and a second nucleic acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 75; or 15) a first nucleic acid molecule comprising DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 82 and a second c acid molecule comprising DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 83.
14. The nucleotide composition of claim 13, wherein 1) DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 7 is shown as SEQ ID NO: 8; 2) DNA ng a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 11 is shown as SEQ ID NO: 12; 3) DNA encoding a light chain variable region (VL) as ented by an amino acid sequence of SEQ ID NO: 15 is shown as SEQ ID NO: 16; 4) DNA ng a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 19 is shown as SEQ ID NO: 20; 5) DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 46 is shown as SEQ ID NO: 47; 6) DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 74 is shown as SEQ ID NO: 76; 7) DNA encoding a light chain variable region (VL) as represented by an amino acid sequence of SEQ ID NO: 82 is shown as SEQ ID NO: 72; 8) DNA ng a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 23 is shown as SEQ ID NO: 24; 9) DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 27 is shown as SEQ ID NO: 28; 10) DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 31 is shown as SEQ ID NO: 32; 11) DNA encoding a heavy chain variable region (VH) as represented by an amino acid ce of SEQ ID NO: 48 is shown as SEQ ID NO: 49; 12) DNA encoding a heavy chain le region (VH) as represented by an amino acid sequence of SEQ ID NO: 75 is shown as SEQ ID NO: 77; and/or 13) DNA encoding a heavy chain variable region (VH) as represented by an amino acid sequence of SEQ ID NO: 83 is shown as SEQ ID NO: 73.
15. The tide composition of any one of claims 12 to 14, comprising: 1) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9 and a second nucleic acid molecule sing DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25; 2) a first nucleic acid molecule sing DNA ng a light chain as represented by an amino acid sequence of SEQ ID NO: 9 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29; 3) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 9 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33; 4) a first nucleic acid le comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 13 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25; 5) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 13 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29; 6) a first nucleic acid molecule comprising DNA encoding a light chain as ented by an amino acid sequence of SEQ ID NO: 13 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid ce of SEQ ID NO: 33; 7) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 and a second nucleic acid le comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25; 8) a first nucleic acid le comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid ce of SEQ ID NO: 29; 9) a first c acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 and a second nucleic acid molecule comprising DNA ng a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33; 10) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25; 11) a first nucleic acid molecule sing DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid molecule comprising DNA ng a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29; 12) a first nucleic acid le comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 21 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33; 13) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 37 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 35; 14) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid ce of SEQ ID NO: 39 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 35; 15) a first c acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 50 and a second c acid molecule sing DNA encoding a heavy chain as represented by an amino acid ce of SEQ ID NO: 52; or 16) a first nucleic acid molecule comprising DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 54 and a second nucleic acid molecule comprising DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 56.
16. The nucleotide composition of claim 15, wherein 1) DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 13 is shown as SEQ ID NO: 14; 2) DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 17 is shown as SEQ ID NO: 18; 3) DNA encoding a light chain as ented by an amino acid ce of SEQ ID NO: 21 is shown as SEQ ID NO: 22; 4) DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 37 is shown as SEQ ID NO: 38; 5) DNA ng a light chain as represented by an amino acid sequence of SEQ ID NO: 39 is shown as SEQ ID NO: 40; 6) DNA encoding a light chain as represented by an amino acid ce of SEQ ID NO: 50 is shown as SEQ ID NO: 51; 7) DNA encoding a light chain as represented by an amino acid sequence of SEQ ID NO: 54 is shown as SEQ ID NO: 55; 8) DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 25 is shown as SEQ ID NO: 26; 9) DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 29 is shown as SEQ ID NO: 30; 10) DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 33 is shown as SEQ ID NO: 34; 11) DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 35 is shown as SEQ ID NO: 36; 12) DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 52 is shown as SEQ ID NO: 53; and/or 13) DNA encoding a heavy chain as represented by an amino acid sequence of SEQ ID NO: 56 is shown as SEQ ID NO: 57.
17. A vector, comprising the nucleotide ition of any one of claims 12 to 16.
18. The vector of claim 17, being a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.
19. A host cell comprising the vector of claim 17 or 18.
20. The host cell of claim 19, wherein the host cell comprising the vector is obtained by vector transformation.
21. The host cell of claims 19 or 20, being bacteria, yeast or mammalian cell.
22. The host cell of any one of claims 19 to 21, being escherichia coli, pichia yeast, Chinese hamster ovary cells or human embryonic kidney 293 cells.
23. A method of ing the isolated antibody or n-binding nt thereof of any one of claims 1 to 11, comprising expressing the antibody or antigen-binding fragment thereof in the host cell of any one of claims 19 to 21 and isolating the antibody or antigen-binding fragment thereof.
24. A pharmaceutical composition comprising a therapeutically effective amount of the isolated antibody or antigen-binding nt thereof of any one of claims 1 to 11, and a pharmaceutical acceptable excipient.
25. A reagent for detecting LIF in biological samples comprising the isolated dy or antigen-binding fragment thereof of any one of claims 1 to 11.
26. The reagent of claim 25, wherein the biological s are blood, serum, urine, biopsy materials, tumor, or any tissues suspected of having al LIF levels.
27. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 11, and/or the ceutical composition of claim 24 in manufacture of a medicament used for inhibiting the expression of LIF and/or blocking the activity of
28. An ex vivo method for detecting the LIF in biological samples comprising (i) obtaining a subject’s tissue or liquid sample, (ii) exposing the tissue or liquid sample to the isolated antibody or antigen-binding nt thereof of any one of claims 1 to 11 or the reagent of claim 25 or 26; and (iii) comparing the LIF binding to the tissue or liquid sample of (ii) with the LIF binding to a control sample, wherein the increase in the amount of the bound LIF compared with the control sample shows the abnormal level of LIF production, expression or activation .
29. The method of claim 28, wherein the tissue or liquid sample comprises blood, serum, urine, biopsy materials, tumor, or any tissues suspected of having al LIF levels.
30. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 11, and/or the pharmaceutical composition of claim 24 in manufacture of a medicament for treating a disease or condition related to LIF.
31. The use of claim 30, wherein the disease or condition related to LIF is tumor.
32. The use of claim 31, wherein the tumor is solid tumor.
33. The use of claim 32, wherein the solid tumor comprises glioblastoma, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer or te cancer. The absorption values The absorption values Log antibody concentration (μg/mL) Log antibody concentration (μg/mL)
NZ788350A 2019-09-29 2020-09-28 Binding molecule specific for LIF and use thereof NZ788350A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNPCT/CN2019/108904 2019-09-29
CNPCT/CN2020/077049 2020-02-27

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NZ788350A true NZ788350A (en) 2022-05-27

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