Polypeptide acting as a light chain and antibody containing the same

Abstract

Polypeptides are provided for use as antibody light chains or light chain variable regions. These polypeptides can be used to construct antibodies with antibody heavy chains or antibody heavy chain variable regions that have binding affinity for different targets (antigens), and the antibodies retain their binding affinity and biological activity for the targets (antigens). Furthermore, antibodies comprising these polypeptides are provided.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This patent application claims the benefit of priority to Chinese Patent Application No. 202310116909.2, filed on February 25, 2023, the entire contents of which are incorporated herein by reference for all purposes.

[0002] The present invention relates to the field of biomedicine, and in particular to a peptide, its use as an antibody light chain, and to a monoclonal or multispecific antibody comprising this peptide as a common light chain. [Background technology]

[0003] Antibodies typically exist as one or more Y-shaped monomers, each composed of four polypeptide chains: two heavy chains and two light chains. The two heavy chains and two light chains of an antibody may be identical, and such an antibody may be called a "homodimer," which has binding affinity and specificity for one target, e.g., an antigen. Alternatively, the two heavy chains and two light chains of an antibody may be different, and such an antibody may be called a "heterodimer," which has binding affinity and specificity for two targets, e.g., antigens. An additional target-binding domain may also be incorporated into the Y-shaped monomer structure.

[0004] It is generally believed that the binding affinity and specificity of an antibody to a target, such as an antigen, are jointly determined by the six complementarity-determining regions (CDRs) in the paired heavy and light chains. However, it has been proposed to provide a polypeptide that can function as a common light chain that can pair with any heavy chain that has binding affinity and specificity for a target, thereby constructing an antibody (homodimer). Based on this, this polypeptide can also function as a common light chain that can pair with two or more antibody heavy chains, thereby constructing a bispecific antibody or even a multispecific antibody (heterodimer, heteromultimer) that has binding affinity and specificity for different targets.

[0005] Furthermore, current conventional methods for producing bispecific antibodies often require the simultaneous transfection of four plasmids to express two different heavy chains and two different light chains. However, when these four polypeptide chains assemble into an antibody, the problem of mispairing between the heavy and light chains can occur. By providing the above-mentioned polypeptide as a common light chain that pairs with two different heavy chains, this problem can be avoided. Summary of the Invention

[0006] To address the above problems, an object of the present disclosure is to provide a polypeptide that can be used as an antibody light chain variable region to construct an antibody by pairing it with an antibody heavy chain or heavy chain variable region specific for a particular target (antigen) or with two or more antibody heavy chain or heavy chain variable regions specific for different targets (antigens). Another object of the present disclosure is to provide an antibody comprising the polypeptide as a light chain variable region. Such an antibody may be, for example, in the form of a homodimer or a heterodimer.

[0007] The present disclosure provides the following technical solutions:

[0008] In a first aspect, the present disclosure provides a polypeptide, the amino acid sequence of which is (1) the amino acid sequence shown in SEQ ID NO: 25, the amino acid sequence shown in SEQ ID NO: 26, and the amino acid sequence shown in SEQ ID NO: 32; (2) the amino acid sequence shown in SEQ ID NO: 31, the amino acid sequence shown in SEQ ID NO: 33, and the amino acid sequence shown in SEQ ID NO: 32, or (3) the amino acid sequence shown in SEQ ID NO: 52, the amino acid sequence shown in SEQ ID NO: 26, and the amino acid sequence shown in SEQ ID NO: 53; The present invention provides a polypeptide comprising:

[0009] Furthermore, the polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:41. SEQ ID NO:2: QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK SEQ ID NO: 13: QIVLTQSPAIMSASPGEKVTMTC SATSGVSYMH WYQQKSGTSPKRWIY DTSRLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK SEQ ID NO: 15: DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIK SEQ ID NO: 16: DIQITQSPSSLSASVGDRVTITC SASSSVSYMH WYQQKPGKAPKRLIY DTSKLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIK SEQ ID NO:41: DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIK

[0010] Furthermore, the polypeptide may comprise the amino acid sequence set forth in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:43. SEQ ID NO:35: QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:36: QIVLTQSPAIMSASPGEKVTMTC SATSGVSYMH WYQQKSGTSPKRWIY DTSRLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:37: DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:38: DIQITQSPSSLSASVGDRVTITC SASSSVSYMH WYQQKPGKAPKRLIY DTSKLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:43: DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0011] Polypeptides functioning as antibody light chains or antibody light chain variable regions provided by the present disclosure can be paired with antibody heavy chains or antibody heavy chain variable regions having binding affinity for different targets (antigens) to construct antibodies with binding affinity for different targets (antigens). For example, a polypeptide functioning as an antibody light chain or antibody light chain variable region can be paired with an antibody heavy chain or antibody heavy chain variable region having binding affinity for a particular target (antigen) to construct an antibody with binding affinity for the target (antigen), where the antibody is a homodimeric antibody having two identical heavy chains and two identical light chains (i.e., a monospecific antibody). Alternatively, for example, a polypeptide functioning as an antibody light chain or antibody light chain variable region can be paired with two antibody heavy chains or antibody heavy chain variable regions each having binding affinity for two targets (antigens), to construct an antibody with binding affinity for two targets (antigens), where the antibody is a heterodimeric antibody having two different heavy chains and two identical light chains (i.e., a bispecific antibody).

[0012] In a second aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a polypeptide provided in the first aspect of the disclosure as an antibody light chain or antibody light chain variable region.

[0013] In the context of this disclosure, an antigen-binding fragment refers to any functional fragment of an antibody that is capable of specifically binding to a target. In the context of this disclosure, the terms "target" and "antigen" are used interchangeably.

[0014] The antibody or antigen-binding fragment thereof provided by the present disclosure may be an IgG-like antibody. In the present disclosure, "IgG-like antibody" means that the antibody or antigen-binding fragment thereof has at least two Fab arms and an optional Fc region and has a conventional monoclonal antibody (mAb) structure or a similar structure. Thus, the antibody or antigen-binding fragment thereof provided by the present disclosure may comprise, in at least one Fab arm, an amino acid sequence set forth in any one of (1) to (3) of the first aspect of the present disclosure as a light chain CDR, or may comprise the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 41 as a light chain variable region (VL). Alternatively, the antibody or antigen-binding fragment thereof provided by the present disclosure may comprise, as a light chain, the amino acid sequence set forth in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 43.

[0015] Furthermore, the antibody or antigen-binding fragment thereof may further comprise, in at least one Fab arm thereof, a heavy chain variable region (VH) that, together with the light chain variable region, forms a binding domain for a target. According to specific embodiments of the present invention, the target may be programmed cell death factor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), programmed cell death 1 ligand 1 (PDL1), or epidermal growth factor receptor (EGFR).

[0016] According to specific embodiments of the invention, the antibody or antigen-binding fragment thereof may comprise, in at least one Fab arm thereof, heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3), respectively, as follows: (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (4) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (5) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (6) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (7) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 48, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 49, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 50, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (8) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 48, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 51, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 50, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 52, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 53; (9) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 54, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 56, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (10) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 48, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 51, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 50, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32, or (11) HCDR1 having the amino acid sequence shown in SEQ ID NO: 54, HCDR2 having the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 56, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 52, LCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 53.

[0017] Furthermore, the antibody or antigen-binding fragment thereof may comprise, in at least one of its Fab arms, each of: (1) the amino acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 2; (2) the amino acid sequence shown in SEQ ID NO: 10 and the amino acid sequence shown in SEQ ID NO: 2; (3) the amino acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 13; (4) the amino acid sequence shown in SEQ ID NO: 10 and the amino acid sequence shown in SEQ ID NO: 13; (5) the amino acid sequence shown in SEQ ID NO: 14 and the amino acid sequence shown in SEQ ID NO: 15; (6) the amino acid sequence shown in SEQ ID NO: 19 and the amino acid sequence shown in SEQ ID NO: 15; (7) the amino acid sequence shown in SEQ ID NO: 14 and the amino acid sequence shown in SEQ ID NO: 16; (8) the amino acid sequence shown in SEQ ID NO: 19 and the amino acid sequence shown in SEQ ID NO: 16; (9) the amino acid sequence shown in SEQ ID NO: 39 and the amino acid sequence shown in SEQ ID NO: 2; (10) The amino acid sequence shown in SEQ ID NO: 40 and the amino acid sequence shown in SEQ ID NO: 41; (11) The amino acid sequence shown in SEQ ID NO: 44 and the amino acid sequence shown in SEQ ID NO: 13; (12) The amino acid sequence shown in SEQ ID NO: 45 and the amino acid sequence shown in SEQ ID NO: 15; (13) The amino acid sequence shown in SEQ ID NO: 40 and the amino acid sequence shown in SEQ ID NO: 15, or (14) The amino acid sequence shown in SEQ ID NO: 45 and the amino acid sequence shown in SEQ ID NO: 41; and a light chain variable region comprising:

[0018] The antibody or antigen-binding fragment thereof may be a murine antibody, chimeric antibody, or humanized antibody, and may further comprise a heavy chain constant region and a light chain constant region. According to specific embodiments of the present invention, the heavy chain constant region may be an IgG1 or IgG4 heavy chain constant region, and the light chain constant region may be a κ light chain constant region or a λ light chain constant region. For example, the heavy chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 17 (hIgG4) or SEQ ID NO: 42 (hIgG1), and the light chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 18 (hκ). Alternatively, the heavy chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 20 (hIgG4, knob) or SEQ ID NO: 21 (hIgG4, hole), and the light chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 18. Alternatively, the heavy chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 46 (hIgG1, knob) or SEQ ID NO: 47 (hIgG1, hole), and the light chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 18.

[0019] At least two Fab arms of an antibody or antigen-binding fragment thereof provided by the present disclosure may be identical, thereby binding to the same target (antigen). In this case, the antibody or antigen-binding fragment thereof may comprise, in its at least two Fab arms, a combination of identical amino acid sequences selected from any one of (1) to (11) above for HCDR1 to HCDR3 and LCDR1 to LCDR3, or a combination of identical amino acid sequences selected from any one of (1) to (14) above for the heavy chain variable region and the light chain variable region. The antibody or antigen-binding fragment thereof may further comprise a heavy chain constant region and a light chain constant region. As described above, for example, the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 42, and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 18. Furthermore, the antibody or antigen-binding fragment thereof may have a heavy chain and a light chain, e.g., it is a monoclonal antibody having two identical heavy chains and two identical light chains.

[0020] Alternatively, at least two Fab arms of an antibody or antigen-binding fragment thereof provided by the present disclosure may be different, thereby binding to different targets (antigens). In this case, the antibody or antigen-binding fragment thereof may comprise, in its at least two Fab arms, a combination of different amino acid sequences selected from any one of (1) to (11) above for HCDR1 to HCDR3 and LCDR1 to LCDR3, or a combination of different amino acid sequences selected from any one of (1) to (14) above for the heavy chain variable region and the light chain variable region. In this case, it is preferred that at least two Fab arms of the antibody or antigen-binding fragment thereof comprise the same light chain CDR sequence or the same light chain variable sequence. For example, both of the at least two Fab arms comprise (1) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32, (2) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32, or (3) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53. Further, for example, both of the at least two Fab arms comprise (1) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2, (2) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13, (3) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, (4) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16, or (5) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41.

[0021] According to a specific embodiment of the invention, the antibody or antigen-binding fragment thereof comprises: (1) In one Fab arm, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 28, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 30, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 33, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 32; and In the other Fab arm, it comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32, or (2) In one Fab arm, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 28, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 30, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 32; and In the other Fab arm, it comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32, or (3) In one Fab arm, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 54, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 55, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 56, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32; and In the other Fab arm, it comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 51, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50, and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32, or (4) In one Fab arm, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 54, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 55, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 56, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 53; and The other Fab arm may comprise: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 48, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 51, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 50, as well as an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 52, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 26, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 53.

[0022] According to specific embodiments of the present invention, the antibody or antigen-binding fragment thereof further comprises: (1) In one Fab arm: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15; and In the other Fab arm: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, or (2) In one Fab arm: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16; and In the other Fab arm: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16, or (3) In one Fab arm: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15; and In the other Fab arm: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15; or (4) In one Fab arm: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41; and In the other Fab arm: it may comprise a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:40 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:41.

[0023] In this case, the antibody or antigen-binding fragment thereof may comprise a heavy chain constant region and a light chain constant region. For example, the heavy chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21, and the light chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 18. Alternatively, the heavy chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 46 (hIgG1, knob) or SEQ ID NO: 47 (hIgG1, hole), and the light chain constant region of the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 18.

[0024] Furthermore, the antibody may be a bispecific antibody, having two different heavy chains but comprising two identical light chains. According to specific embodiments of the invention, the bispecific antibody may comprise as a light chain the amino acid sequence set forth in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 43.

[0025] According to specific embodiments of the present invention, bispecific antibodies provided by the present disclosure are shown in Tables 9 and 36 in the "Detailed Description of the Invention" section.

[0026] The bispecific antibodies shown in Table 9 comprise the following domains:

[0027] The bispecific antibodies KA-1835 H28G27V4×H107D1210-hIgG4M and KA-1836 H107D1210×H28G27V4-hIgG4M each contain two Fab arms targeting PD1 and LAG3, respectively: one Fab arm contains a heavy chain variable region targeting PD1 (SEQ ID NO: 14) paired with a light chain variable region (SEQ ID NO: 15), and the other Fab arm contains a heavy chain variable region targeting LAG3 (SEQ ID NO: 19) paired with a light chain variable region (SEQ ID NO: 15). This bispecific antibody contains two identical light chains (SEQ ID NO: 37).

[0028] The bispecific antibody KA-1793 H28G27V4xH107D1210-hIgG4M contains two Fab arms targeting PD1 and LAG3, respectively: one Fab arm contains a heavy chain variable region targeting PD1 (SEQ ID NO: 14) paired with a light chain variable region (SEQ ID NO: 16), and the other Fab arm contains a heavy chain variable region targeting LAG3 (SEQ ID NO: 19) paired with a light chain variable region (SEQ ID NO: 16). This bispecific antibody contains two identical light chains (SEQ ID NO: 38).

[0029] The bispecific antibodies shown in Table 36 comprise the following domains:

[0030] The bispecific antibody KA-2072 H22A12×58E3-CLC01 contains two Fab arms targeting EGFR and PDL1, respectively: one Fab arm contains a heavy chain variable region targeting EGFR (SEQ ID NO: 45) paired with a light chain variable region (SEQ ID NO: 15), and the other Fab arm contains a heavy chain variable region targeting PDL1 (SEQ ID NO: 40) paired with a light chain variable region (SEQ ID NO: 15). This bispecific antibody contains two identical light chains (SEQ ID NO: 37).

[0031] The bispecific antibody KA-2073 H22A12x58E3-CLC02 contains two Fab arms targeting EGFR and PDL1, respectively: one Fab arm contains a heavy chain variable region targeting EGFR (SEQ ID NO: 45) paired with a light chain variable region (SEQ ID NO: 41), and the other Fab arm contains a heavy chain variable region targeting PDL1 (SEQ ID NO: 40) paired with a light chain variable region (SEQ ID NO: 41). This bispecific antibody contains two identical light chains (SEQ ID NO: 43).

[0032] The present disclosure provides polypeptides, and experiments have shown that a polypeptide that functions as an antibody light chain or an antibody light chain variable region can be paired with an antibody heavy chain or an antibody heavy chain variable region that has binding affinity for a different target (antigen) to construct an antibody with binding affinity and biological activity for the different target (antigen).

[0033] In a third aspect, the present disclosure provides the use of the polypeptide described in the first aspect in constructing an antibody. As described above, the antibody may be a homodimeric antibody having two identical heavy chains and two identical light chains, or a heterodimeric antibody having two different heavy chains and two identical light chains. The polypeptide may be used as the light chain or light chain variable region of the antibody.

[0034] In a fourth aspect, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide described in the first aspect.

[0035] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 shows the results of detecting the binding of hybridoma cell culture supernatant containing anti-LAG3 antibody to LAG3-expressing cells. [Figure 2] FIG. 1 shows the results of detecting the blocking of LAG3 binding to tumor cells by hybridoma cell culture supernatants containing anti-LAG3 antibodies. [Figure 3] FIG. 1 shows the results of detecting the binding of hybridoma cell culture supernatant containing anti-PD1 antibody to PD1-expressing cells. [Figure 4] FIG. 1 shows the results of detecting the blocking of PD1 binding to PDL1-expressing cells by hybridoma cell culture supernatants containing anti-PD1 antibodies. [Figure 5] FIG. 1 shows the results of detecting the binding of anti-PD1 humanized antibody and anti-PD1 chimeric antibody to PD1-expressing cells. [Figure 6] FIG. 1 shows the results of detecting the blocking of PD1 binding to PDL1-expressing cells by anti-PD1 humanized antibodies and anti-PD1 chimeric antibodies. [Figure 7] FIG. 1 shows the results of detecting the binding of anti-LAG3 humanized antibody and anti-LAG3 chimeric antibody to LAG3-expressing cells. [Figure 8] FIG. 1 shows the results of detecting the blocking of LAG3 binding to tumor cells by anti-LAG3 humanized antibodies and anti-LAG3 chimeric antibodies. [Figure 9] FIG. 1 shows the results of detecting the binding of PD1×LAG3 bispecific antibodies to PD1-expressing cells. [Figure 10] FIG. 1 shows the results of detecting the binding of PD1×LAG3 bispecific antibodies to LAG3-expressing cells. [Figure 11] FIG. 1 shows the results of detecting the blocking of PD1 binding to PDL1-expressing cells by the PD1×LAG3 bispecific antibody. [Figure 12] FIG. 1 shows the results of detecting the blocking of LAG3 binding to tumor cells by the PD1×LAG3 bispecific antibody. [Figure 13] FIG. 1 shows the results of detecting the binding of PD1×LAG3 bispecific antibodies to PD1-expressing cells. [Figure 14] FIG. 1 shows the results of detecting the binding of PD1×LAG3 bispecific antibodies to LAG3-expressing cells. [Figure 15] FIG. 1 shows the results of detecting the blocking of PD1 binding to PDL1-expressing cells by the PD1×LAG3 bispecific antibody. [Figure 16] FIG. 1 shows the results of detecting the blocking of LAG3 binding to tumor cells by the PD1×LAG3 bispecific antibody. [Figure 17] 17A shows the results of detecting the binding of hybridoma cell culture supernatant containing an anti-PDL1 antibody to PDL1-expressing cells, where 17A is a positive control antibody and 17B is the hybridoma cell culture supernatant. [Figure 18] 18A shows the results of detecting the blocking of PDL1 binding to PD1-expressing cells by hybridoma cell culture supernatant containing anti-PDL1 antibody, where 18A is a positive control antibody and 18B is the hybridoma cell culture supernatant. [Figure 19] FIG. 1 shows the results of detecting blockade of PDL1 / PD1 signaling by anti-PDL1 mouse antibodies. [Figure 20] FIG. 1 shows the results of detecting the binding of anti-PDL1 humanized antibodies to PDL1-expressing cells. [Figure 21] FIG. 1 shows the results of detecting the blocking of PDL1 binding to PD1-expressing cells by anti-PDL1 humanized antibodies. [Figure 22] FIG. 1 shows the results of detecting the blockade of PDL1 / PD1 signaling by anti-PDL1 humanized antibodies. [Figure 23] 23A shows the results of detecting the binding of hybridoma cell culture supernatant containing an anti-EGFR antibody to EGFR-expressing cells, where 23A is a positive control antibody and 23B is the hybridoma cell culture supernatant. [Figure 24]1 shows the results of detecting the blocking of EGFR-Fc binding to EGF by anti-EGFR mouse antibodies, where 24A is a positive control antibody and 24B is an anti-EGFR mouse antibody. [Figure 25] FIG. 1 shows the results of detecting blockade of EGFR signaling by anti-EGFR mouse antibodies. [Figure 26] FIG. 1 shows the results of detecting the binding of anti-EGFR humanized antibodies to EGFR-expressing cells. [Figure 27] FIG. 1 shows the results of detecting the blocking of the binding of EGFR-Fc to EGF by anti-EGFR humanized antibodies. [Figure 28] FIG. 1 shows the results of detecting the blockade of EGFR signaling by anti-EGFR humanized antibodies. [Figure 29] FIG. 1 shows the results of detecting the binding of EGFR×PDL1 bispecific antibodies to PDL1-expressing cells. [Figure 30] FIG. 1 shows the results of detecting the blockade of PDL1 binding to PD1-expressing cells by an EGFR×PDL1 bispecific antibody. [Figure 31] FIG. 1 shows the results of detecting the blockade of PDL1 / PD1 signaling by an EGFR×PDL1 bispecific antibody. [Figure 32] FIG. 1 shows the results of detecting the binding of EGFR×PDL1 bispecific antibodies to EGFR-expressing cells. [Figure 33] FIG. 1 shows the results of detecting the blockade of EGF binding to EGFR-expressing cells by an EGFR×PDL1 bispecific antibody. [Figure 34] FIG. 1 shows the results of detecting the blockade of EGFR signaling by an EGFR×PDL1 bispecific antibody. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention will now be described with reference to specific examples, which will be understood by those skilled in the art as being merely illustrative of the present invention and are not intended to limit the scope of the present invention in any way.

[0038] All experimental procedures in the following examples are conventional unless otherwise specified. All raw materials and reagents used in the following examples are commercially available unless otherwise specified.

[0039] Example 1 Screening for common light chains Prior art analysis revealed that germline sequences such as KV4 are more frequently expressed in mice and pair with mouse heavy chains at a higher rate. Therefore, we selected germline sequences from this category, including the following mouse germline sequence, KV4-59, which is relatively easy to humanize, has good solubility, low immunogenicity, and does not have potential modification sites.

[0040] >KV4-59 (SEQ ID NO: 1): QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPP FGSGTKLEIK

[0041] This sequence was modified. Because the light chain CDR3 plays an important role in the binding affinity of the antibody to the antigen, amino acids were added after the CDR3 (see underlined part) to improve the affinity to the potential antigen. The following three sequences were obtained:

[0042] >KV4-59-1 (SEQ ID NO: 2): QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK >KV4-59-2 (SEQ ID NO: 3): QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPWT FGSGTKLEIK >KV4-59-3 (SEQ ID NO: 4): QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPFT FGSGTKLEIK

[0043] Each of the above three sequences was used as the antibody light chain variable region sequence and paired with the heavy chain variable region sequence of a mouse anti-chicken lysozyme antibody as follows, and a chimeric antibody was constructed using human heavy chain constant regions and human light chain constant regions.

[0044] >Sequence of the heavy chain variable region of mouse anti-chicken lysozyme antibody (SEQ ID NO: 5): LDVKLQESGPGLVKPSQSLSLTCSVTGYSFTSGYYWNWIRLFPGNKLEWMGYVSYFGTNNYNPSLKNRFSITRDTSKNQFFLKLNSVSTEDTATYYCARGEEWDWSFDVWGTGTTVTVSS

[0045] By detecting the properties of the three chimeric antibodies, such as expression level, thermal stability, solubility, and freeze-thaw stability, it was confirmed that the constructed antibodies had excellent properties when KV4-59-1 (SEQ ID NO: 2) was used as the antibody light chain variable region sequence.

[0046] Example 2 Construction of transgenic mice Based on the antibody lambda light chain gene sequence of C57 mice, sgRNAs were designed from both ends of the gene cluster. Using the Cas9-gRNA Target Efficiency Detection Kit (Vazyme, catalog number: VK007-30T), the following sgRNA sequences, which showed high cleavage efficiency, were selected through in vitro validation: TGAATGCCATGTACTTATGG (SEQ ID NO: 6) and AAGTTCAGCTCCTAAAATGG (SEQ ID NO: 7). The sgRNA sequences were synthesized and microinjected into mouse fertilized eggs along with spCas91.1 protein. The sgRNA cleaved both ends of the target gene fragment via the Cas9 protein, generating a double-stranded DNA break. The genomic ends were repaired via the non-homologous end joining (NHEJ) pathway, resulting in deletion of the middle target fragment and knockout of the target gene. Two weeks after birth, mice were genotyped to confirm lambda gene knockout.

[0047] sgRNAs were designed based on the mouse antibody kappa light chain sequence. Using a Cas9-gRNA Target Efficiency Detection Kit (Vazyme, catalog number: VK007-30T), the following sgRNA sequences were selected through in vitro validation, demonstrating high cleavage efficiency: IGK-L1: GTGAATGCCATGTACTTATGG (SEQ ID NO: 8), IGK-R1: CAAGTTCAGCTCCTAAAATGG (SEQ ID NO: 9). The sgRNA sequences were synthesized and microinjected into mouse fertilized eggs along with spCas91.1 protein. The sgRNA cleaved at the target site via Cas9 protein, creating a nick. Using a donor DNA fragment with homologous arms and the target fragment KV4-59-1 (SEQ ID NO: 2), knock-in of the DNA fragment at the target site was achieved via homologous recombination. Two weeks after birth, mice were genotyped to confirm knock-in of the common light chain gene.

[0048] Heterozygous mice with a lambda chain knockout were mated with heterozygous mice with a common light chain knockin. Two weeks after birth, each offspring was genotyped to detect the presence of the common light chain knockin and the lambda chain knockout status. This process was continued until mice homozygous for the common light chain knockin and both lambda and kappa chain knockout were obtained.

[0049] Example 3: Preparation of hybridoma cells secreting anti-LAG3 antibodies The mice constructed in Example 2 were immunized with a fusion protein (referred to as "LAG3-mFc") of the human LAG3 extracellular domain (NCBI reference sequence: NP_002277.4) and mouse IgG2a-Fc (Genbank accession number AAH31470.1). Mice with higher titers were selected, and serum was collected from them. The mice were then sacrificed, their spleens were removed, and splenocytes were isolated. The splenocytes were fused with cultured myeloma cells to obtain hybridoma cells.

[0050] An ELISA plate was coated with a fusion protein (LAG3-hFc) of the human LAG3 extracellular domain and human IgG1-Fc (Genbank accession number CAC20454.1). The binding activity of hybridoma cell culture supernatants to this fusion protein was detected by ELISA, and numerous positive hybridoma cell lines secreting anti-LAG3 antibodies were obtained.

[0051] Example 4 Detection of binding of hybridoma cell culture supernatant containing anti-LAG3 antibody to LAG3-expressing cells The gene sequence encoding human LAG3 (NCBI reference sequence: NM_002286.6) was cloned into a PLVX packaging vector (Clontech, virus package mix, catalog number: 631275), and the recombinant plasmid was transfected into 293T cells. Resistant cell lines were then selected using puromycin to obtain 293T cells stably expressing human LAG3 (referred to as "293T-LAG3 cells"; Kyinno Biotechnology Co. Ltd. (referred to as "Kyinno"), catalog number: KC-0214). 293T-LAG3 cells were then cultured at 1 × 10 per mL in PBS containing 2% FBS. 7 The cells were prepared as a cell suspension at a concentration of 1000 cells.

[0052] 50 μL of the cell suspension was added to one flow cytometry tube (sample tube), followed by 50 μL of either the hybridoma cell culture supernatant to be detected or the control antibody 28F6-7, followed by incubation at 4°C for 60 minutes. Next, 1 mL of flow cytometry buffer was added to each flow cytometry tube, which was then centrifuged at 1200 rpm for 5 minutes and the supernatant was discarded. This washing step was repeated three times. Simultaneously, control tube 1 (cell suspension only, without culture supernatant or secondary antibody (described below)) and control tube 2 (cell suspension and secondary antibody only, without culture supernatant) were prepared.

[0053] To each flow cytometry tube, 100 μL of flow cytometry buffer was added to resuspend the cells, followed by the addition of 5 μL of PE-conjugated anti-mouse IgG Fc secondary antibody (PE anti-mouse IgG Fc, Biolegend, catalog number: 409304), followed by incubation at 4°C for 30 minutes in the dark. Next, 1 mL of flow cytometry buffer was added to each tube, which was then centrifuged at 1200 rpm for 5 minutes at room temperature, and the supernatant was discarded. This washing step was repeated three times. To each flow cytometry tube, 250 μL of flow cytometry buffer was added to resuspend the cells, and the cells were thoroughly mixed and detected. The detection results are shown in Table 1 and Figure 1.

[0054] Table 1. Results of detection of binding of hybridoma cell culture supernatant containing anti-LAG3 antibody to LAG3-expressing cells [Table 1]

[0055] Note: The control antibody 28F6-7 is a previously validated anti-LAG3 hybridoma antibody.

[0056] Example 5 Detection of blocking of LAG3 binding to tumor cells by hybridoma cell culture supernatant containing anti-LAG3 antibody Daudi cells (Kyinno, Cat. No. KC-0271) were harvested, washed once with FACS buffer, and plated at 2 × 10 cells per well in a 96-deep-well plate. 5 pieces~5×10 5Cells were plated at 1000 x g / well. 50 μL of hybridoma cell culture supernatant or control antibody 28F6-7 (diluted 3.16-fold from a starting concentration of 20 μg / mL) and 500 ng of the fusion protein LAG3-hFc were added to the plate as a premix per well, followed by incubation for 2 hours. Next, 400 μL of FACS buffer was added per well, followed by washing twice. PE-conjugated anti-human IgG Fc secondary antibody (PE anti-human IgG Fc, SouthernBiotech, catalog number: 2010-09) was added, followed by incubation for 1 hour. After washing twice again with 400 μL of FACS buffer per well, the cells were detected. The detection results are shown in Table 2 and Figure 2.

[0057] Table 2. Detection results of blocking of LAG3 binding to tumor cells by hybridoma cell culture supernatant containing anti-LAG3 antibody [Table 2]

[0058] The murine antibody was obtained from hybridoma cell line KD-0019 28G2-7 and designated "28G2." Its variable region sequences are as follows (heavy and light chain CDRs are underlined and identified according to the KABAT numbering scheme; ibid. for subsequent cases):

[0059] Heavy chain variable region of anti-LAG3 murine antibody 28G2 (SEQ ID NO: 10; HCDR: SEQ ID NO: 22 / 23 / 24): DVKLQESGPGLVKPSQSLSLTCSVTGYSFT SGYYWN WIRLFPGNKLEWMG YVSYFGTNNYNPSLKN RFSITRDTSKNQFFLKLNSVSTEDTATYYCAR GEEWDWSFDV WGTGTTVTVSS

[0060] Light chain variable region of anti-LAG3 murine antibody 28G2 (SEQ ID NO: 11; LCDR: SEQ ID NOs: 25 / 26 / 27): DIVLTQSPAFMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPTRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSTNPPYT FGGGTKLEIK

[0061] Example 6: Preparation of hybridoma cells secreting anti-PD1 antibodies The mice constructed in Example 2 were immunized with a fusion protein (referred to as "PD1-mFc") of the human PD1 extracellular domain (NCBI reference sequence: NP_005009.2) and mouse IgG2a-Fc (Genbank accession number AAH31470.1). Mice with the highest titers were selected, and serum was collected from them. The mice were then sacrificed, their spleens were removed, and splenocytes were isolated. The splenocytes were fused with cultured myeloma cells to obtain hybridoma cells.

[0062] ELISA plates were coated with a fusion protein (PD1-hFc) of the human PD1 extracellular domain and human IgG1-Fc (Genbank accession number CAC20454.1). The binding activity of hybridoma cell culture supernatants to this fusion protein was detected by ELISA, and numerous positive hybridoma cell lines secreting anti-PD1 antibodies were obtained.

[0063] Example 7 Detection of binding of hybridoma cell culture supernatant containing anti-PD1 antibody to PD1-expressing cells The gene sequence encoding human PD1 (NCBI reference sequence: NM_005018.3) was cloned into the PLVX packaging vector (Clontech, viral packaging mix, catalog number: 631275). The recombinant plasmid was transfected into 293T cells, and resistant cell lines were selected using puromycin to obtain 293T cells stably expressing human PD1 (referred to as "293T-PD1 cells"; Kyinno, catalog number: KC-0204). 293T-PD1 cells were then cultured at 1 × 10 per mL in PBS containing 2% FBS. 7 The cells were prepared as a cell suspension at a concentration of 1000 cells.

[0064] 50 μL of the cell suspension was added to one flow cytometry tube (sample tube), followed by 50 μL of the hybridoma cell culture supernatant to be detected, followed by incubation at 4°C for 60 minutes. Next, 1 mL of flow cytometry buffer was added to each flow cytometry tube, which was then centrifuged at 1200 rpm for 5 minutes and the supernatant was discarded. This washing process was repeated three times. Simultaneously, control tube 1 (cell suspension only, without culture supernatant or secondary antibody (described below)) and control tube 2 (cell suspension and secondary antibody only, without culture supernatant) were prepared.

[0065] To each flow cytometry tube, 100 μL of flow cytometry buffer was added to resuspend the cells, followed by the addition of 5 μL of PE-conjugated anti-mouse IgG Fc secondary antibody (Biolegend, catalog number: 409304), followed by incubation at 4°C for 30 minutes in the dark. Next, 1 mL of flow cytometry buffer was added to each tube, which was then centrifuged at 1200 rpm for 5 minutes at room temperature, and the supernatant was discarded. This washing step was repeated three times. To each flow cytometry tube, 250 μL of flow cytometry buffer was added to resuspend the cells, and the cells were thoroughly mixed and detected. The detection results are shown in Table 3 and Figure 3.

[0066] Table 3. Detection results of binding of hybridoma cell culture supernatant containing anti-PD1 antibody to PD1-expressing cells [Table 3]

[0067] Example 8 Detection of blocking of PD1 binding to PDL1-expressing cells by hybridoma cell culture supernatant containing anti-PD1 antibody 293T cells overexpressing human PDL1 (referred to as "293T-PDL1 cells"; Kyinno, Cat. No.: KC-0205) were harvested, washed once with FACS buffer, and plated at 2 × 10 cells per well in a 96-deep-well plate.5 pieces~5×10 5 Cells were plated at 1000 x g / well. 50 μL of hybridoma cell culture supernatant and 500 ng of human PD1-hFc (NCBI reference sequence: NP_005009.2) were added per well as a premix, followed by incubation for 2 hours. Next, 400 μL of FACS buffer was added per well, followed by washing twice. PE-conjugated anti-human IgG Fc secondary antibody (SouthernBiotech, catalog number: 2010-09) was added, followed by incubation for 1 hour. After washing twice again with 400 μL of FACS buffer per well, cells were detected. The detection results are shown in Table 4 and Figure 4.

[0068] Table 4. Detection results of blocking of PD1 binding to PDL1-expressing cells by hybridoma cell culture supernatant containing anti-PD1 antibody [Table 4]

[0069] The murine antibody was obtained from hybridoma cell line KD-22-0054 107D12-10 and named "107D12-10". Its variable region sequences are as follows (heavy and light chain CDRs are underlined):

[0070] Heavy chain variable region of anti-PD1 mouse antibody 107D12-10 (SEQ ID NO: 12; HCDR: SEQ ID NOs: 28 / 29 / 30): QVQLKESGGGLVKPGGSLKLSCAASGFTFS DYGMH WVRQAPEKGLEWVA YISSGSYTIYYADTVRD RFTISRDSAKNTLFLQMTSLRSEDTAMYYCVR RGPGYFGSTALDY WGQGTTLTVSS

[0071] Light chain variable region of anti-PD1 mouse antibody 107D12-10 (SEQ ID NO: 13; LCDR: SEQ ID NOs: 31 / 33 / 32): QIVLTQSPAIMSASPGEKVTMTC SATSGVSYMH WYQQKSGTSPKRWIY DTSRLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK

[0072] Example 9 Construction of anti-PD1 humanized antibody and anti-PD1 chimeric antibody The heavy and light chain variable regions of the anti-PD1 murine antibody 107D12-10 were humanized to obtain a humanized antibody sequence. The coding sequence of the humanized antibody sequence was linked to the coding sequences of the human IgG4 heavy chain constant region (SEQ ID NO: 17) and the human kappa light chain constant region (SEQ ID NO: 18), respectively. The resulting coding gene was cloned into a eukaryotic expression vector and expressed to obtain an anti-PD1 humanized antibody designated "KA-1807 H107D12-10V4-hIgG4M." The variable region sequences (SEQ ID NO: 14 and SEQ ID NO: 15) and full-length light chain (SEQ ID NO: 37) are shown below (heavy and light chain CDRs are underlined).

[0073] In addition, two chimeric antibodies were constructed based on the murine antibody 107D12-10. One chimeric antibody was constructed as follows: the coding sequence of the heavy chain variable region of murine antibody 107D12-10 (SEQ ID NO: 12) was linked to the coding sequence of the human IgG4 heavy chain constant region, and the coding sequence of the light chain variable region of murine antibody 107D12-10 (SEQ ID NO: 13) was linked to the coding sequence of the human kappa light chain constant region. The resulting coding genes were cloned into a eukaryotic expression vector and expressed to obtain an anti-PD-1 chimeric antibody designated "KA-1773 M107D12-10CLC1-hIgG4M." The variable region sequences (SEQ ID NO: 12 and SEQ ID NO: 13) and full-length light chain sequence (SEQ ID NO: 36) are shown below. Another chimeric antibody was constructed as follows: the coding sequence for the heavy chain variable region of murine antibody 107D12-10 (SEQ ID NO: 12) was linked to the coding sequence for the human IgG4 heavy chain constant region, and the coding sequence for the light chain variable region shown in SEQ ID NO: 2 (KV4-59-1) was linked to the coding sequence for the human kappa light chain constant region. The resulting coding gene was cloned into a eukaryotic expression vector and expressed to obtain an anti-PD-1 chimeric antibody designated "KA-1774 M107D12-10CLC2-hIgG4M." Its variable region sequences (SEQ ID NO: 12 and SEQ ID NO: 2) and full-length light chain sequence (SEQ ID NO: 35) are shown below.

[0074] Heavy chain variable region of anti-PD1 humanized antibody KA-1807 H107D12-10V4-hIgG4M (SEQ ID NO: 14; HCDR: SEQ ID NOs: 28 / 29 / 30): QVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMH WIRQAPGKGLEWVA YISSGSYTIYYADTVRD RFTISRDSAKNSLYLQMNSLRAEDTAVYYCVR RGPGYFGSTALDY WGQGTTVTVSS

[0075] >Light chain variable region of anti-PD1 humanized antibody KA-1807 H107D12-10V4-hIgG4M (SEQ ID NO: 15; LCDR: SEQ ID NOs: 31 / 33 / 32): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIK

[0076] Heavy chain variable region of anti-PD1 mouse antibody 107D12-10 (SEQ ID NO: 12; HCDR: SEQ ID NOs: 28 / 29 / 30): QVQLKESGGGLVKPGGSLKLSCAASGFTFS DYGMH WVRQAPEKGLEWVA YISSGSYTIYYADTVRD RFTISRDSAKNTLFLQMTSLRSEDTAMYYCVR RGPGYFGSTALDY WGQGTTLTVSS

[0077] Light chain variable region of anti-PD1 mouse antibody 107D12-10 (SEQ ID NO: 13; LCDR: SEQ ID NOs: 31 / 33 / 32): QIVLTQSPAIMSASPGEKVTMTC SATSGVSYMH WYQQKSGTSPKRWIY DTSRLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK

[0078] >KV4-59-1 (SEQ ID NO: 2; LCDR: SEQ ID NO: 25 / 26 / 32): QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK

[0079] Human heavy chain CH1 and Fc sequence (SEQ ID NO: 17): hIgG4-S228P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0080] >Human light chain CL sequence (SEQ ID NO: 18): hκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0081] >Light chain (SEQ ID NO: 35): QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0082] >Light chain (SEQ ID NO: 36): QIVLTQSPAIMSASPGEKVTMTC SATSGVSYMH WYQQKSGTSPKRWIY DTSRLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0083] >Light chain (SEQ ID NO: 37): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0084] Example 10: Detection of binding of anti-PD1 humanized antibody and anti-PD1 chimeric antibody to PD1-expressing cells Flow cytometry (FACS) was used to detect the binding of the humanized and chimeric antibodies to PD1-expressing cells. The experimental procedures were the same as those described in Example 7, except that nivolumab and pembrolizumab were used as positive control antibodies and a goat anti-human IgG-PE secondary antibody (SouthernBiotech, catalog number: 2010-09) was added. The detection results are shown in Table 5 and Figure 5.

[0085] Table 5. Detection results of binding of anti-PD1 humanized antibody and anti-PD1 chimeric antibody to PD1-expressing cells [Table 5]

[0086] Example 11 Detection of blocking of PD1 binding to PDL1-expressing cells by anti-PD1 humanized antibody and anti-PD1 chimeric antibody 293T-PDL1 cells were harvested, washed once with FACS buffer, and plated at 2 × 10 cells per well in a 96-deep-well plate. 5 pieces~5×10 5 Cells were plated at 1000 x g. 50 μL of one of the target antibodies (anti-PD1 chimeric antibodies KA-1773 M107D12-10CLC1-hIgG4M and KA-1774 M107D12-10CLC2-hIgG4M, humanized antibody KA-1807 H107D12-10V4-hIgG4M, and control antibodies nivolumab and pembrolizumab) and 30 ng of the fusion protein PD1-mFc were added to the plate as a premix per well, followed by incubation for 2 hours. Next, 400 μL of FACS buffer was added per well, followed by washing twice. PE-conjugated anti-mouse IgG Fc secondary antibody was added, followed by incubation for 1 hour. After washing twice again with 400 μL of FACS buffer per well, the cells were detected. The detection results are shown in Table 6 and Figure 6.

[0087] Table 6. Detection results of blocking of PD1 binding to PDL1-expressing cells by anti-PD1 antibodies [Table 6]

[0088] Example 12 Construction of anti-LAG3 humanized antibody and anti-LAG3 chimeric antibody The heavy chain variable region of the anti-LAG3 murine antibody 28G2 was humanized to obtain the humanized antibody sequence (SEQ ID NO: 19). Furthermore, the light chain variable region (SEQ ID NO: 15) of the anti-PD1 humanized antibody obtained in Example 9 was used as the light chain variable region to construct an anti-LAG3 humanized antibody. The coding sequence of the humanized antibody sequence was linked to the coding sequences of the human IgG4 heavy chain constant region (SEQ ID NO: 17) and the human kappa light chain constant region (SEQ ID NO: 18), respectively. The resulting coding genes were cloned into a eukaryotic expression vector and expressed to obtain anti-LAG3 humanized antibodies designated "KA-1780 H28G2-7V2-hIgG4M" and "KA-1782 H28G2-7V4-hIgG4M." The variable region sequences (SEQ ID NO: 19 and SEQ ID NO: 15) and full-length light chain (SEQ ID NO: 37) of antibody KA-1782 H28G2-7V4-hIgG4M are shown below.

[0089] Furthermore, two chimeric antibodies were constructed based on the murine antibody 28G2 and the murine antibody 107D12-10. One chimeric antibody was constructed as follows: the coding sequence of the heavy chain variable region of the murine antibody 28G2 (SEQ ID NO: 10) was linked to the coding sequence of the human IgG4 heavy chain constant region, and the coding sequence of the light chain variable region of the murine antibody 107D12-10 (SEQ ID NO: 13) was linked to the coding sequence of the human kappa light chain constant region. The resulting coding genes were cloned into a eukaryotic expression vector and expressed to obtain an anti-LAG3 chimeric antibody designated "KA-1728 M28G2-7CLC1-hIgG4M." The variable region sequences (SEQ ID NO: 10 and SEQ ID NO: 13) and full-length light chain sequence (SEQ ID NO: 36) are shown below. Another chimeric antibody was constructed as follows: the coding sequence of the heavy chain variable region of murine antibody 28G2 (SEQ ID NO: 10) was linked to the coding sequence of the human IgG4 heavy chain constant region, and the coding sequence of the light chain variable region shown in SEQ ID NO: 2 (KV4-59-1) was linked to the coding sequence of the human kappa light chain constant region. The resulting coding gene was cloned into a eukaryotic expression vector and expressed to obtain an anti-LAG3 chimeric antibody designated "KA-1733 M28G2-7CLC2-hIgG4M." Its variable region sequences (SEQ ID NO: 10 and SEQ ID NO: 2) and full-length light chain sequence (SEQ ID NO: 35) are shown below.

[0090] Heavy chain variable region of anti-LAG3 humanized antibody KA-1782 H28G2-7V4-hIgG4M (SEQ ID NO: 19; HCDR: SEQ ID NOs: 22 / 34 / 24): QVQLQESGPGLVKPSETLSLTTCTVSGYSFT SGYYWN WIRQPPGKGLEWIG YVSYFGTANYNPALKN RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR GEEWDWSFDV WGQGTTVTVSS

[0091] >Light chain variable region of anti-LAG3 humanized antibody KA-1782 H28G2-7V4-hIgG4M (light chain variable region of anti-PD1 humanized antibody KA-1807 H107D12-10V4-hIgG4M; SEQ ID NO: 15; LCDR: SEQ ID NOs: 31 / 33 / 32): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIK

[0092] Heavy chain variable region of anti-LAG3 murine antibody 28G2 (SEQ ID NO: 10; HCDR: SEQ ID NO: 22 / 23 / 24): DVKLQESGPGLVKPSQSLSLTCSVTGYSFT SGYYWN WIRLFPGNKLEWMG YVSYFGTNNYNPSLKN RFSITRDTSKNQFFLKLNSVSTEDTATYYCAR GEEWDWSFDV WGTGTTVTVSS

[0093] Light chain variable region of anti-PD1 mouse antibody 107D12-10 (SEQ ID NO: 13; LCDR: SEQ ID NOs: 31 / 33 / 32): QIVLTQSPAIMSASPGEKVTMTC SATSGVSYMH WYQQKSGTSPKRWIY DTSRLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK

[0094] >KV4-59-1 (SEQ ID NO: 2; LCDR: SEQ ID NO: 25 / 26 / 32): QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK

[0095] Human heavy chain CH1 and Fc sequence (SEQ ID NO: 17): hIgG4-S228P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0096] >Human light chain CL sequence (SEQ ID NO: 18): hκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0097] >Light chain (SEQ ID NO: 35): QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0098] >Light chain (SEQ ID NO: 36): QIVLTQSPAIMSASPGEKVTMTC SATSGVSYMH WYQQKSGTSPKRWIY DTSRLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0099] >Light chain (SEQ ID NO: 37): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0100] Example 13 Detection of binding of anti-LAG3 humanized antibody and anti-LAG3 chimeric antibody to LAG3-expressing cells Flow cytometry (FACS) was used to detect the binding of the humanized and chimeric antibodies obtained in Example 12 to LAG3-expressing cells. The experimental procedure was the same as that described in Example 4, except that leratolimab was used as a positive control antibody and a goat anti-human IgG-PE secondary antibody (SouthernBiotech, catalog number: 2010-09) was added. The detection results are shown in Table 7 and Figure 7.

[0101] Table 7. Results of detection of binding of anti-LAG3 humanized antibody and anti-LAG3 chimeric antibody to LAG3-expressing cells [Table 7]

[0102] Example 14 Detection of blocking of LAG3 binding to tumor cells by anti-LAG3 humanized antibody and anti-LAG3 chimeric antibody Daudi cells were harvested, washed once with FACS buffer, and plated at 2 × 10 cells per well in a 96-deep-well plate. 5 pieces~5×10 5 Cells were plated at 1000 x g. 50 μL of one of the antibodies to be detected (chimeric antibodies KA-1728 M28G2-7CLC1-hIgG4M and KA-1733 M28G2-7CLC2-hIgG4M, humanized antibodies KA-1780 H28G2-7V2-hIgG4M and KA-1782 H28G2-7V4-hIgG4M, and control antibody leratolimab) and 500 ng of human LAG3-mFc were added as a premix per well of the plate, followed by incubation for 2 hours. Next, 400 μL of FACS buffer was added per well and the plate was washed twice. PE-conjugated anti-mouse IgG Fc secondary antibody was then added and incubated for 1 hour. After washing twice again with 400 μL of FACS buffer per well, the cells were detected. The detection results are shown in Table 8 and Figure 8.

[0103] Table 8. Detection results of blocking of LAG3 binding to tumor cells by anti-LAG3 humanized antibody and anti-LAG3 chimeric antibody [Table 8]

[0104] Example 15 Construction of PD1 x LAG3 Bispecific Antibody The coding sequence for the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16 (see below, sequences obtained by humanizing KV4-59-1) was ligated to the coding sequence for the human kappa light chain constant region (SEQ ID NO: 18), thereby obtaining common light chain 1 (SEQ ID NO: 37) or common light chain 2 (SEQ ID NO: 38) for use in constructing the bispecific antibody.

[0105] The coding sequence for the heavy chain variable region of the anti-PD1 humanized antibody was linked to the coding sequence for the human heavy chain CH1 and Fc sequences ("knob") below, and the coding sequence for the heavy chain variable region of the anti-LAG3 humanized antibody was linked to the coding sequence for the human heavy chain CH1 and Fc sequences ("hole") below. Alternatively, the coding sequence for the heavy chain variable region of the anti-LAG3 humanized antibody was linked to the coding sequence for the human heavy chain CH1 and Fc sequences ("knob") below, and the coding sequence for the heavy chain variable region of the anti-PD1 humanized antibody was linked to the coding sequence for the human heavy chain CH1 and Fc sequences ("hole") below.

[0106] The resulting coding genes were cloned into a eukaryotic expression vector, resulting in three recombinant expression plasmids (one encoding common light chain 1 or common light chain 2, one encoding the PD1-targeting heavy chain, and one encoding the LAG3-targeting heavy chain). The three plasmids were transiently co-transfected using the HEK293F expression system to produce the PD1 x LAG3 bispecific antibody. Briefly, for a 1 L shaker flask, HEK293F cells were cultured at 1 x 10 per mL in 250 mL of culture medium. 6 The cells were seeded at 2 × 10 cells per mL and incubated at 110 rpm in 5% CO2. The next day, the three expression plasmids prepared in advance were mixed with the transfection reagent at a specific ratio, and the transfection complex was prepared at 2 × 10 cells per mL. 6 The cells were added to a cell density of 1000 cells, and after 24 hours, nutrients and DNA inhibitors were replenished. After 5-7 days of cell culture, the expression supernatant was collected, centrifuged, filtered, and purified using a MabSelectSure affinity chromatography column (GE Healthcare). The purity of the purified antibody was determined by SDS-PAGE electrophoresis, and the antibody concentration was determined using a Nanodrop.

[0107] The domains contained in the resulting PD1×LAG3 bispecific antibody are shown in Table 9.

[0108] Table 9. Bispecific antibodies containing a common light chain [Table 9]

[0109] Heavy chain variable region of anti-PD1 humanized antibody KA-1807 H107D12-10V4-hIgG4M (SEQ ID NO: 14; HCDR: SEQ ID NOs: 28 / 29 / 30): QVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGMH WIRQAPGKGLEWVA YISSGSYTIYYADTVRD RFTISRDSAKNSLYLQMNSLRAEDTAVYYCVR RGPGYFGSTALDY WGQGTTVTVSS

[0110] Heavy chain variable region of anti-LAG3 humanized antibody KA-1782 H28G2-7V4-hIgG4M (SEQ ID NO: 19; HCDR: SEQ ID NOs: 22 / 34 / 24): QVQLQESGPGLVKPSETLSLTTCTVSGYSFT SCIENTIFIC WIRQPPGKGLEWIG YVSYFGTANYNPALKN RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR GEEWDWSFDV WGQGTTVTVSS

[0111] >Light chain variable region of anti-PD1 humanized antibody KA-1807 H107D12-10V4-hIgG4M (SEQ ID NO: 15; LCDR: SEQ ID NOs: 31 / 33 / 32): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIK

[0112] >Humanized sequence of KV4-59-1 (SEQ ID NO: 16; LCDR: SEQ ID NOs: 25 / 26 / 32): DIQITQSPSSLSASVGDRVTITC SASSSVSYMH WYQQKPGKAPKRLIY DTSKLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIK

[0113] Human heavy chain CH1 and Fc sequence ("knob"; hIgG4; SEQ ID NO: 20): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0114] Human heavy chain CH1 and Fc sequence ("hole"; hIgG4; SEQ ID NO: 21): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0115] >Human light chain CL sequence (SEQ ID NO: 18): hκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0116] >Light chain (SEQ ID NO: 37): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0117] >Light chain (SEQ ID NO: 38): DIQITQSPSSLSASVGDRVTITC SASSSVSYMH WYQQKPGKAPKRLIY DTSKLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0118] Example 16 Detection of binding of PD1 x LAG3 bispecific antibodies with a common light chain 1 to PD1- and LAG3-expressing cells Binding of the PD1×LAG3 bispecific antibody to PD1-expressing cells (293T-PD1 cells) and LAG3-expressing cells (293T-LAG3 cells) was detected using flow cytometry (FACS) using 293T-PD1 cells and 293T-LAG3 cells, respectively. The experimental procedures were as described in Examples 4 and 7, respectively. The detection results are shown in Table 10 and Figure 9 (target protein: PD1), and Table 11 and Figure 10 (target protein: LAG3).

[0119] Table 10. Detection results of binding of PD1×LAG3 bispecific antibodies to PD1-expressing cells [Table 10]

[0120] Table 11. Detection results of binding of PD1×LAG3 bispecific antibodies to LAG3-expressing cells [Table 11]

[0121] Example 17 Detection of blocking of PD1 binding to PDL1-expressing cells and LAG3 binding to tumor cells by PD1 x LAG3 bispecific antibodies with a common light chain 1 Blockade of PD1 binding to PDL1-expressing cells and LAG3 binding to tumor cells by the PD1×LAG3 bispecific antibody was detected using 293T-PDL1 cells and Daudi cells, respectively, by flow cytometry (FACS). The experimental procedures were as described in Example 5 and Example 8, respectively. The detection results are shown in Table 12 and Figure 11 (target protein: PD1), and Table 13 and Figure 12 (target protein: LAG3).

[0122] Table 12. Detection results of blocking of PD1 binding to PDL1-expressing cells by PD1×LAG3 bispecific antibody [Table 12]

[0123] Table 13. Detection results of blocking LAG3 binding to tumor cells by PD1×LAG3 bispecific antibody [Table 13]

[0124] Example 18: Detection of the affinity of PD1 x LAG3 bispecific antibodies with a common light chain 1 to PD1 and LAG3 The affinity of the PD1×LAG3 bispecific antibody for PD1 and LAG3 was detected using a ForteBio Octet instrument. The mobile phase consisted of PD1-His (Kyinno, catalog number: KP-1023) diluted to concentrations of 400 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, and LAG3-His (ACRO, catalog number: LA3-H5222) diluted to concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, which were simultaneously detected. The results are shown in Tables 14 and 15.

[0125] Table 14. Affinity of PD1×LAG3 bispecific antibodies to PD1 [Table 14]

[0126] Table 15. Affinity of PD1 x LAG3 bispecific antibodies to LAG3 [Table 15]

[0127] Example 19 Detection of binding of PD1 x LAG3 bispecific antibodies with a common light chain 2 to PD1- and LAG3-expressing cells Binding of the PD1×LAG3 bispecific antibody to PD1-expressing cells (293T-PD1 cells) and LAG3-expressing cells (293T-LAG3 cells) was detected using flow cytometry (FACS) using 293T-PD1 cells and 293T-LAG3 cells, respectively. The experimental procedures were as described in Examples 4 and 7, respectively. The detection results are shown in Table 16 and Figure 13 (target protein: PD1), and Table 17 and Figure 14 (target protein: LAG3).

[0128] Table 16. Detection results of binding of PD1×LAG3 bispecific antibodies to PD1-expressing cells [Table 16]

[0129] Table 17. Detection results of binding of PD1 x LAG3 bispecific antibodies to LAG3-expressing cells [Table 17]

[0130] Example 20: Detection of blocking of PD1 binding to PDL1-expressing cells and LAG3 binding to tumor cells by PD1 x LAG3 bispecific antibodies with common light chain 2 Blockade of PD1 binding to PDL1-expressing cells and LAG3 binding to tumor cells by the PD1×LAG3 bispecific antibody was detected using 293T-PDL1 cells and Daudi cells, respectively, by flow cytometry (FACS). The experimental procedures were as described in Example 5 and Example 8, respectively. The detection results are shown in Table 18 and Figure 15 (target protein: PD1), and Table 19 and Figure 16 (target protein: LAG3).

[0131] Table 18. Detection results of blocking of PD1 binding to PDL1-expressing cells by PD1×LAG3 bispecific antibody [Table 18]

[0132] Table 19. Detection results of blocking LAG3 binding to tumor cells by PD1×LAG3 bispecific antibody [Table 19]

[0133] Example 21: Detection of the affinity of PD1 x LAG3 bispecific antibodies with a common light chain 2 for PD1 and LAG3 The affinity of the PD1×LAG3 bispecific antibody for PD1 and LAG3 was detected using a ForteBio Octet instrument. The mobile phase consisted of PD1-His (Kyinno, catalog number: KP-1023) diluted to concentrations of 400 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, and LAG3-His (ACRO, catalog number: LA3-H5222) diluted to concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, which were simultaneously detected. The results are shown in Tables 20 and 21.

[0134] Table 20. Affinity of PD1×LAG3 bispecific antibodies to PD1 [Table 20]

[0135] Table 21. Affinity of PD1 x LAG3 bispecific antibodies to LAG3 [Table 21]

[0136] Example 22: Preparation of hybridoma cells secreting anti-PDL1 antibodies The mice constructed in Example 2 were immunized with a fusion protein of human PDL1 extracellular domain and mouse Fc (referred to as "PDL1-mFc"; Kyinno, catalog number: KP-1008). The experimental procedures were the same as those described in Example 3. Mice with higher titers were selected, and serum was collected therefrom. The mice were then dissected, their spleens were removed, and splenocytes were isolated. The splenocytes were fused with cultured myeloma cells to obtain hybridoma cells.

[0137] ELISA plates were coated with PDL1-his, and the binding activity of hybridoma cell culture supernatants to PDL1-his was detected by ELISA. A number of positive hybridoma cell lines secreting anti-PDL1 antibodies were obtained.

[0138] Example 23: Detection of binding of hybridoma cell culture supernatant containing anti-PDL1 antibody to PDL1-expressing cells 293T-PDL1 cells overexpressing human PDL1 (Kyinno, Cat. No.: KC-0205) were cultured at 1 × 10 per mL in PBS containing 2% FBS. 7 The cells were prepared as a cell suspension at a concentration of 1000 cells.

[0139] Flow cytometry was used to detect binding of hybridoma cell culture supernatant containing anti-PDL1 antibody to 293T-PDL1 cells. The experimental procedure was the same as that described in Example 4, except that atezolizumab (Kyinno, catalog number: KA-1303-02) was used as a positive control, and 5 μL of PE-conjugated anti-mouse IgG Fc secondary antibody was added to detect binding of the hybridoma cell culture supernatant, and goat anti-human IgG-PE secondary antibody (SouthernBiotech, catalog number: 2010-09) was added to detect binding of the positive control antibody atezolizumab. The detection results are shown in Table 22 and Figure 17.

[0140] Table 22: Results of detection of binding of hybridoma cell culture supernatant containing anti-PDL1 antibody to PDL1-expressing cells [Table 22]

[0141] Example 24 Detection of blocking of PDL1-mFc binding to PD1-expressing cells by hybridoma cell culture supernatant containing anti-PDL1 antibody 293T cells overexpressing human PD1 (referred to as "293T-PD1 cells"; Kyinno, catalog number: KC-0204) were washed once with FACS buffer and plated at 2 × 10 per well in a 96-deep-well plate. 5 pieces~5×10 5 Cells were plated at 1000 x g / well. 50 μL of hybridoma cell culture supernatant or positive control antibody atezolizumab and 500 ng of human PDL1-mFc (Kyinno, catalog number: KP-1008) or PDL1-hFc (Kyinno, catalog number: KP-1009) (NCBI reference sequence: NP_054862.1) were added as a premix per well to the plate, followed by incubation for 2 hours. Next, 400 μL of FACS buffer was added per well and the plate was washed twice. Then, goat anti-human IgG-PE secondary antibody (SouthernBiotech, catalog number: 2010-09) or PE-conjugated anti-mouse IgG Fc secondary antibody (Biolegend, catalog number: 405307) was added, followed by incubation for 1 hour. After washing twice again with 400 μL of FACS buffer per well, the cells were detected. The detection results are shown in Table 23 and Figure 18.

[0142] Table 23. Detection results of blocking of PDL1 binding to PD1-expressing cells by hybridoma cell culture supernatant containing anti-PDL1 antibody [Table 23]

[0143] The murine antibody was obtained from hybridoma cell line KD-22-0227 58E3-2 and designated "58E3-2." Its variable region sequences are as follows (heavy and light chain CDRs are underlined and identified according to the KABAT numbering scheme):

[0144] Heavy chain variable region of anti-PDL1 mouse antibody 58E3-2 (SEQ ID NO: 39; HCDR: SEQ ID NO: 48 / 49 / 50): QVQLKESGGGLVKPGGSLKLSCAASGFTFS BRING IT WVRQAPEKGLEWVA FISSGSSTIYYADTVKG RFTISRDNAKNTLFLQMTSLRSEDTAMYYCTR RDFIGSADAMDY WGQGTSVTVSS

[0145] >Light chain variable region of anti-PDL1 mouse antibody 58E3-2 (KV4-59-1; SEQ ID NO: 2; LCDR: SEQ ID NO: 25 / 26 / 32): QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK

[0146] Example 25 Detection of blockade of PDL1 / PD1 signaling by anti-PDL1 mouse antibodies Jurkat NFAT-PD1-luc cells (Kyinno, catalog number: KC-1503, expressing luciferase upon NFAT signaling activation) and 293T OS8-hPDL1 cells (Kyinno, catalog number: KC-1148, expressing surface-bound OKT3 antibody) were adjusted to a cell density of 20,000 cells per 45 μL in RPMI-1640 + 10% FBS medium, and 45 μL of each cell type was added per well to a 96-well plate. Ten μL of serially diluted positive control antibody (atezolizumab) or one of the anti-PDL1 mouse antibodies was added per well, followed by co-culture for 6 hours. Subsequently, 50 μL of Bright-Lite solution (Vazyme, catalog number: DD1204) was added to each well to detect luciferase activity. Wells without antibody served as background activation controls. The fold increase in activation (over background) was plotted against antibody concentration, and the IC for anti-PDL1 antibodies was calculated. 50 The values ​​were calculated, and the detection results are shown in Table 24 and Figure 19.

[0147] Table 24. Detection results of blockade of PDL1 / PD1 signaling by anti-PDL1 mouse antibody [Table 24]

[0148] Example 26: Detection of affinity of anti-PDL1 mouse antibodies to PDL1 The affinity of the anti-PDL1 mouse antibody to PDL1 was detected using a ForteBio Octet instrument. The mobile phase was PDL1-His (Kyinno, Cat. No. KP-1022) diluted to concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM. The results are shown in Table 25.

[0149] Table 25. Affinity of anti-PDL1 mouse antibodies to PDL1 [Table 25]

[0150] Example 27 Construction of anti-PDL1 humanized antibody The heavy and light chain variable regions of the anti-PDL1 murine antibody 58E3-2 were humanized to obtain the humanized antibody sequences (SEQ ID NOs: 40 and 41). The coding sequences of the humanized antibody sequences were linked to the coding sequences of the human IgG1 heavy chain constant region (SEQ ID NO: 42) and the human kappa light chain constant region (SEQ ID NO: 18), respectively. The resulting coding genes were cloned into a eukaryotic expression vector and expressed to obtain an anti-PDL1 humanized antibody designated "KA-2016 H58E3-2V4-2-hIgG1." The variable region sequences (SEQ ID NOs: 40 and 41) and full-length light chain (SEQ ID NO: 43) are shown below (heavy and light chain CDRs are underlined).

[0151] Heavy chain variable region of anti-PDL1 humanized antibody KA-2016 H58E3-2V4-2-hIgG1 (SEQ ID NO: 40; HCDR: SEQ ID NOs: 48 / 51 / 50): QVQLVESGGGLVKPGGSLRLSCAASGFTFS BRING IT WIRQAPGKGLEWVA FISSGSSTIYYADAVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCTR RDFIGSADAMDY WGQGTTVTVSS

[0152] >Light chain variable region of anti-PDL1 humanized antibody KA-2016 H58E3-2V4-2-hIgG1 (SEQ ID NO: 41; LCDR: SEQ ID NOs: 52 / 26 / 53): DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIK

[0153] Human heavy chain CH1 and Fc sequence (SEQ ID NO: 42): hIgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0154] >Human light chain CL sequence (SEQ ID NO: 18): hκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0155] >Light chain (SEQ ID NO: 43): DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0156] Example 28 Detection of binding of anti-PDL1 humanized antibodies to PDL1-expressing cells 293T-PDL1 cells were cultured at 1 × 10 per mL in PBS containing 2% FBS. 7 The cells were prepared as a cell suspension at a concentration of 1000 cells.

[0157] Flow cytometry was used to detect binding of the anti-PDL1 humanized antibody KA-2016 H58E3-2V4-2-hIgG1 or the positive control antibody atezolizumab to 293T-PDL1 cells. The experimental procedure was as described in Example 23. The detection results are shown in Table 26 and Figure 20.

[0158] Table 26. Detection results of binding of anti-PDL1 humanized antibodies to PDL1-expressing cells [Table 26]

[0159] Example 29 Detection of blocking of binding of PDL1-mFc to PD1-expressing cells by anti-PDL1 humanized antibodies Blockade of PDL1-mFc binding to PD1-expressing cells by anti-PDL1 humanized antibodies was detected. The experimental procedure was as described in Example 24, except that 50 μL of serially diluted humanized antibody KA-2016 H58E3-2V4-2-hIgG1 or positive control antibody atezolizumab and 500 ng of human PDL1-mFc were added as a premix per well, and finally co-incubated with a PE-conjugated anti-mouse IgG Fc secondary antibody. The detection results are shown in Table 27 and Figure 21.

[0160] Table 27. Detection results of blocking of PDL1 binding to PD1-expressing cells by anti-PDL1 humanized antibodies [Table 27]

[0161] Example 30 Detection of blockade of PDL1 / PD1 signaling by anti-PDL1 humanized antibodies Blockade of PDL1 / PD1 signaling by anti-PDL1 humanized antibodies was detected. The experimental procedures were as described in Example 25. The detection results are shown in Table 28 and Figure 22.

[0162] Table 28. Detection results of blockade of PDL1 / PD1 signaling by anti-PDL1 humanized antibodies [Table 28]

[0163] Example 31: Preparation of hybridoma cells secreting anti-EGFR antibodies The mice constructed in Example 2 were immunized with a fusion protein of human EGFR extracellular domain (NCBI reference sequence: BC118665.1) and mouse Fc (referred to as "EGFR-mFc"; Kyinno, catalog number: KP-1148). The experimental procedures were as described in Example 3. Mice with higher titers were selected, and serum was collected therefrom. The mice were then dissected, their spleens were removed, and splenocytes were isolated. The splenocytes were fused with cultured myeloma cells to obtain hybridoma cells.

[0164] ELISA plates were coated with EGFR-his (Kyinno, Cat. No. KP-1341). The binding activity of the hybridoma cell culture supernatant to EGFR-his was detected by ELISA, and a large number of positive hybridoma cell lines secreting anti-EGFR antibodies were obtained.

[0165] Example 32 Detection of binding of hybridoma cell culture supernatant containing anti-EGFR antibody to EGFR-expressing cells The gene sequence encoding human EGFR (NCBI reference sequence: BC118665.1) was cloned into the PLVX packaging vector (Clontech, viral packaging mix, catalog number: 631275), and the recombinant plasmid was transfected into CT26 cells. Resistant cell lines were then selected using puromycin to obtain CT26 cells stably expressing human EGFR (referred to as "CT26-EGFR cells"; Kyinno, catalog number: KC-1451). CT26-EGFR cells were then cultured at 1 x 10 per mL in PBS containing 2% FBS. 7 The cells were prepared as a cell suspension at a concentration of 1000 cells.

[0166] Flow cytometry was used to detect the binding of hybridoma cell culture supernatants containing anti-EGFR antibodies to CT26-EGFR cells. The experimental procedure was the same as that described in Example 4, except that cetuximab (Kyinno, Cat. No. KA-1398) was used, and 5 μL of a PE-conjugated anti-mouse IgG Fc secondary antibody was added to detect the binding of the hybridoma cell culture supernatant, and 5 μL of a goat anti-human IgG-PE secondary antibody (SouthernBiotech, Cat. No. 2010-09) was added to detect the binding of the positive control antibody cetuximab. The detection results are shown in Table 29 and Figure 23.

[0167] Table 29: Results of detection of binding of hybridoma cell culture supernatant containing anti-EGFR antibody to EGFR-expressing cells [Table 29]

[0168] The murine antibody was obtained from hybridoma cell line KD-22-0198 22A12-2 and named "22A12-2." Its variable region sequences are as follows (heavy and light chain CDRs are underlined):

[0169] Heavy chain variable region of anti-EGFR mouse antibody 22A12-2 (SEQ ID NO: 44; HCDR: SEQ ID NO: 54 / 55 / 56): EVQLQQSGPELVKPGASVKISKASGYTFT DYMMN WVKQSHGKSLEWIG DINPNDGGTSYNQKFKG KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR EILYYVTSFYFDY WGQGTTLTVSS

[0170] >Light chain variable region of anti-EGFR mouse antibody 22A12-2 (SEQ ID NO: 13; LCDR: SEQ ID NOs: 31 / 33 / 32): QIVLTQSPAIMSASPGEKVTMTC SATSGVSYMH WYQQKSGTSPKRWIY DTSRLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK

[0171] Example 33 Detection of blocking of EGFR-Fc binding to EGF by anti-EGFR mouse antibodies ELISA plates were coated with EGF-His (2 μg / mL, Acro, catalog number: EGF-H52H3) dissolved in PBS overnight at 4°C. The next day, the plates were blocked with blocking buffer (PBS containing 1% BSA) for 1 hour at room temperature. Serially diluted control antibody cetuximab or hybridoma antibodies were co-incubated with 0.4 μg / mL EGFR-mFc (Kyinno, catalog number: KP-1148) or EGFR-hFc (Kyinno, catalog number: KP-1149). The mixture was then added to the EGF-His-coated plate and incubated at room temperature for 1 hour. The plate was then washed with PBS containing 0.5% Tween-20 and subsequently co-incubated with HRP-conjugated anti-mouse IgG secondary antibody (diluted 1:10,000) or HRP-conjugated anti-human IgG secondary antibody (diluted 1:10,000). The plate was then washed again with PBS containing 0.5% Tween-20, and HRP substrate was added for color development. The binding signal was detected and the IC 50 The values ​​were calculated, and the detection results are shown in Table 30 and Figure 24.

[0172] Table 30. Detection of blocking of EGFR binding to EGF by anti-EGFR mouse antibodies [Table 30]

[0173] Example 34 Detection of blockade of EGFR signaling by anti-EGFR mouse antibodies 293T-NFAT-Luc2-EGFR cells (Kyinno, catalog number: KC-2952, which express luciferase upon EGFR signaling activation) were adjusted to a cell density of 20,000 cells per 80 μL in RPMI-1640 + 10% FBS medium. 10 μL of serially diluted positive control antibody cetuximab or one of the anti-EGFR mouse antibodies was added per well of the plate and co-incubated for 30 minutes. After 30 minutes of co-incubation, 10 μL of EGF-His (final concentration: 20 ng / mL, Acro, catalog number: EGF-H52H3) was added per well and cultured overnight. Subsequently, 50 μL of Bright-Lite solution (Vazyme, catalog number: DD1204) was added to each well to detect luciferase activity. Wells without EGF served as background activation controls. The fold increase in activation (over background) was plotted against antibody concentration to determine the IC values ​​for the anti-EGFR antibodies. 50 The values ​​were calculated, and the detection results are shown in Table 31 and Figure 25.

[0174] Table 31. Detection results of blockade of EGFR signaling by anti-EGFR mouse antibodies [Table 31]

[0175] Example 35: Detection of affinity of anti-EGFR mouse antibodies to EGFR The affinity of the anti-EGFR mouse antibodies to EGFR was detected using a ForteBio Octet instrument. The mobile phase was EGFR-His (Kyinno, Cat. No. KP-1150) diluted to concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM. The results are shown in Table 32.

[0176] Table 32. Affinity of anti-EGFR mouse antibodies to EGFR [Table 32]

[0177] Example 36 Construction of anti-EGFR humanized antibody The heavy chain variable region of the anti-EGFR murine antibody 22A12-2 was humanized to obtain the humanized antibody sequence (SEQ ID NO: 45). Furthermore, the light chain variable region (SEQ ID NO: 15) of the anti-PD1 humanized antibody obtained in Example 9 was used as the light chain variable region to construct an anti-EGFR humanized antibody. The coding sequence of the humanized antibody sequence was linked to the coding sequences of the human IgG1 heavy chain constant region (SEQ ID NO: 42) and the human κ light chain constant region (SEQ ID NO: 18), respectively. The resulting coding gene was cloned into a eukaryotic expression vector and expressed to obtain an anti-EGFR humanized antibody designated "KA-1063 H22A12-2V1-hIgG1." The variable region sequences (SEQ ID NO: 45 and SEQ ID NO: 15) and full-length light chain (SEQ ID NO: 37) are shown below (heavy and light chain CDRs are underlined):

[0178] Heavy chain variable region of anti-EGFR humanized antibody KA-1063 H22A12-2V1-hIgG1 (SEQ ID NO: 45; HCDR: SEQ ID NOs: 54 / 55 / 56): QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYMMN WVRQAPGQGLEWIG DINPNDGGTSYNQKFKG RATLTVDKSTSTAYMELSSLRSEDTAVYYCAR EILYYVTSFYFDY WGQGTTVTVSS

[0179] >Light chain variable region of anti-EGFR humanized antibody 1063 H22A12-2V1-hIgG1 (light chain variable region of anti-PD1 humanized antibody KA-1807 H107D12-10V4-hIgG4M; SEQ ID NO: 15; LCDR: SEQ ID NOs: 31 / 33 / 32): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIK

[0180] Human heavy chain CH1 and Fc sequence (SEQ ID NO: 42): hIgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0181] >Human light chain CL sequence (SEQ ID NO: 18): hκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0182] >Light chain (SEQ ID NO: 37): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0183] Example 37 Detection of binding of anti-EGFR humanized antibodies to EGFR-expressing cells CT26-EGFR cells were cultured at 1 × 10 per mL in PBS containing 2% FBS. 7 The cells were prepared as a cell suspension at a concentration of 1000 cells.

[0184] Flow cytometry was used to detect the binding of anti-EGFR humanized antibodies to CT26-EGFR cells. The experimental procedures were as described in Example 32. The detection results are shown in Table 33 and Figure 26.

[0185] Table 33. Detection results of binding of anti-EGFR humanized antibodies to EGFR-expressing cells [Table 33]

[0186] Example 38 Detection of blocking of EGFR-Fc binding to EGF by anti-EGFR humanized antibodies Blockade of EGFR binding to EGF by anti-EGFR humanized antibodies was detected. The experimental procedure was the same as that described in Example 33, except that the control antibody cetuximab and serially diluted humanized antibody KA-1063 H22A12-2V1-hIgG1 were used, which were then co-incubated with 0.4 μg / mL of EGFR-mFc (Kyinno, Cat. No. KP-1148) and finally co-incubated with an HRP-conjugated anti-mouse IgG secondary antibody. The detection results are shown in Table 34 and Figure 27.

[0187] Table 34. Detection results of blocking of EGFR binding to EGF by anti-EGFR humanized antibodies [Table 34]

[0188] Example 39 Detection of blockade of EGFR signaling by anti-EGFR humanized antibodies Blockade of EGFR signaling by anti-EGFR humanized antibodies was detected. The experimental procedures were as described in Example 34. The detection results are shown in Table 35 and Figure 28.

[0189] Table 35. Detection results of EGFR signaling blockade by anti-EGFR humanized antibodies [Table 35]

[0190] Example 40 Construction of EGFR x PDL1 bispecific antibody The coding sequence for the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 41 was linked to the coding sequence for the human kappa light chain constant region (SEQ ID NO: 18), thereby obtaining common light chain 1 (SEQ ID NO: 37) or common light chain 3 (SEQ ID NO: 43) for use in constructing the bispecific antibody.

[0191] The coding sequence for the heavy chain variable region of the anti-EGFR humanized antibody was linked to the coding sequence for the following human heavy chain CH1 and Fc sequences ("knob"), and the coding sequence for the heavy chain variable region of the anti-PDL1 humanized antibody was linked to the coding sequence for the following human heavy chain CH1 and Fc sequences ("hole").

[0192] An EGFR×PDL1 bispecific antibody was produced. The experimental procedure was in accordance with that described in Example 15. The domains contained in the resulting EGFR×PDL1 bispecific antibody are shown in Table 36.

[0193] Table 36. Bispecific antibodies containing common light chain 1 or common light chain 3 [Table 36]

[0194] Heavy chain variable region of anti-EGFR humanized antibody KA-1063 H22A12-2V1-hIgG1 (SEQ ID NO: 45; HCDR: SEQ ID NOs: 54 / 55 / 56): QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYMMN WVRQAPGQGLEWIG DINPNDGGTSYNQKFKG RATLTVDKSTSTAYMELSSLRSEDTAVYYCAR EILYYVTSFYFDY WGQGTTVTVSS

[0195] Human heavy chain CH1 and Fc sequence ("knob"; hIgG1; SEQ ID NO: 46): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0196] Heavy chain variable region of anti-PDL1 humanized antibody KA-2016 H58E3-2V4-2-hIgG1 (SEQ ID NO: 40; HCDR: SEQ ID NOs: 48 / 51 / 50): QVQLVESGGGLVKPGGSLRLSCAASGFTFS BRING IT WIRQAPGKGLEWVA FISSGSSTIYYADAVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCTR RDFIGSADAMDY WGQGTTVTVSS

[0197] Human heavy chain CH1 and Fc sequence ("hole"; hIgG1; SEQ ID NO: 47): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0198] >Light chain variable region of anti-EGFR humanized antibody KA-1063 H22A12-2V1-hIgG1 (light chain variable region of anti-PD1 humanized antibody KA-1807 H107D12-10V4-hIgG4M; SEQ ID NO: 15; LCDR: SEQ ID NOs: 31 / 33 / 32): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIK

[0199] >Light chain (SEQ ID NO: 37): DIQITQSPSSLSASVGDRVTITC SATSGVSYMH WYQQKPGKAPKRLIY DTSRLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0200] >Light chain variable region of anti-PDL1 humanized antibody KA-2016 H58E3-2V4-2-hIgG1 (SEQ ID NO: 41; LCDR: SEQ ID NOs: 52 / 26 / 53): DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIK

[0201] >Light chain (SEQ ID NO: 43): DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0202] Example 41 Detection of binding of EGFR x PDL1 bispecific antibodies with common light chain 1 or common light chain 3 to PDL1-expressing cells Flow cytometry was used to detect the binding of the EGFR×PDL1 bispecific antibody to PDL1-expressing cells. The experimental procedures were as described in Example 28. The detection results are shown in Table 37 and Figure 29.

[0203] Table 37. Results of detection of binding of EGFR x PDL1 bispecific antibodies with common light chain 1 or common light chain 3 to PDL1-expressing cells [Table 37]

[0204] Example 42 Detection of blocking of binding of PDL1-mFc to PD1-expressing cells by EGFR×PDL1 bispecific antibodies with common light chain 1 or common light chain 3 Blockade of PDL1-mFc binding to PD1-expressing cells by EGFRxPDL1 bispecific antibodies was detected. The experimental procedure was as described in Example 29, except that 50 μL of serially diluted EGFRxPDL1 bispecific antibodies KA-2072 H22A12x58E3-CLC01, KA-2073-H22A12x58E3-CLC0, or the positive control antibody atezolizumab and 1.2 μg / mL human PDL1-mFc were added as a premix per well, followed by co-incubation with a PE-conjugated anti-mouse IgG Fc secondary antibody. The detection results are shown in Table 38 and Figure 30.

[0205] Table 38. Detection results of blocking of PDL1 binding to PD1-expressing cells by EGFR x PDL1 bispecific antibody [Table 38]

[0206] Example 43 Detection of Blockade of PDL1 / PD1 Signaling by EGFRxPDL1 Bispecific Antibodies with Common Light Chain 1 or Common Light Chain 3 Blockade of PDL1 / PD1 signaling by EGFR×PDL1 bispecific antibodies was detected. The experimental procedures were as described in Example 30. The detection results are shown in Table 39 and Figure 31.

[0207] Table 39. Detection results of blockade of PDL1 / PD1 signaling by EGFR x PDL1 bispecific antibodies with common light chain 1 or common light chain 3 [Table 39]

[0208] Example 44 Detection of binding of EGFRxPDL1 bispecific antibodies with common light chain 1 or common light chain 3 to EGFR-expressing cells Flow cytometry was used to detect the binding of the EGFRxPDL1 bispecific antibody to CT26-EGFR cells. The experimental procedure was as described in Example 32, except that 5 μL of goat anti-human IgG-PE secondary antibody (SouthernBiotech, Catalog No.: 2010-09) was added. The detection results are shown in Table 40 and Figure 32.

[0209] Table 40. Results of detection of binding of EGFR x PDL1 bispecific antibodies with common light chain 1 or common light chain 3 to EGFR-expressing cells [Table 40]

[0210] Example 45 Detection of blocking of EGF-mFc binding to EGFR-expressing cells by EGFRxPDL1 bispecific antibodies with common light chain 1 or common light chain 3 CT26 cells overexpressing human EGFR, i.e., "CT26-EGFR cells," were collected. EGFRxPDL1 bispecific antibodies KA-2072 H22A12x58E3-CLC01, KA-2073-H22A12x58E3-CLC0, or the positive control antibody cetuximab were serially diluted and co-incubated with CT26-EGFR cells, followed by co-incubation with 0.05 μg / mL EGF-mFc (Acro, catalog number: EGF-H525b) and finally with a PE-conjugated anti-mouse IgG Fc secondary antibody (Biolegend, catalog number: 405307). Binding of EGF-mFc to the cells was detected using flow cytometry. The detection results are shown in Table 41 and Figure 33.

[0211] Table 41. Detection results of blocking of EGF binding to EGFR-expressing cells by EGFR x PDL1 bispecific antibodies with common light chain 1 or common light chain 3 [Table 41]

[0212] Example 46 Detection of blockade of EGFR signaling by EGFRxPDL1 bispecific antibodies with common light chain 1 or common light chain 3 Blockade of EGFR signaling by the EGFR×PDL1 bispecific antibody was detected. The experimental procedures were as described in Example 34. The detection results are shown in Table 42 and Figure 34.

[0213] Table 42. Results of detection of blockade of EGFR signaling by EGFR x PDL1 bispecific antibodies with common light chain 1 or common light chain 3 [Table 42]

[0214] Example 47: Detection of the affinity of EGFR×PDL1 bispecific antibodies with common light chain 1 or common light chain 3 to EGFR and PDL1 The affinity of the EGFR×PDL1 bispecific antibody to EGFR and PDL1 was detected. The experimental procedures were as described in Example 26 and Example 35. The detection results are shown in Table 43.

[0215] Table 43. Results of detection of affinity for EGFR and PDL1 of EGFR x PDL1 bispecific antibodies having common light chain 1 or common light chain 3 [Table 43]

[0216] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes and modifications to the present invention without departing from the spirit of the present invention, which should be included in the scope of the appended claims.

Claims

1. A polypeptide having an amino acid sequence: (1) the amino acid sequence shown in SEQ ID NO: 25, the amino acid sequence shown in SEQ ID NO: 26, and the amino acid sequence shown in SEQ ID NO: 32, or (2) the amino acid sequence shown in SEQ ID NO: 31, the amino acid sequence shown in SEQ ID NO: 33, and the amino acid sequence shown in SEQ ID NO: 32, or (3) the amino acid sequence shown in SEQ ID NO: 52, the amino acid sequence shown in SEQ ID NO: 26, and the amino acid sequence shown in SEQ ID NO: 53; A polypeptide comprising:

2. The polypeptide of claim 1, characterized in that the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:

41.

3. The polypeptide of claim 1 or 2, characterized in that the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO:

43.

4. An antibody or antigen-binding fragment thereof comprising the polypeptide defined in any one of claims 1 to 3 as an antibody light chain or light chain variable region, Preferably, the antibody or antigen-binding fragment thereof is an IgG-like antibody having at least two Fab arms and optionally an Fc region.

5. The antibody or antigen-binding fragment thereof comprises, in at least one Fab arm thereof, an amino acid sequence defined in any one of (1) to (3) of claim 1 as a light chain CDR, or an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 41 as a light chain variable region, or The antibody or antigen-binding fragment thereof of claim 4, characterized in that the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 43 as a light chain.

6. The antibody or antigen-binding fragment thereof comprises, in at least one Fab arm thereof, heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3), respectively, as follows: (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (4) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (5) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (6) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (7) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 48, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 49, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 50, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (8) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 48, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 51, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 50, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 52, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 53; (9) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 54, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 56, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32; (10) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 48, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 51, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 50, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32, or (11) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 54, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 56, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 52, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 53; The antibody or antigen-binding fragment thereof according to claim 4 or 5, characterized in that:

7. The antibody or antigen-binding fragment thereof comprises, in at least one of its Fab arms, (1) the amino acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 2; (2) the amino acid sequence shown in SEQ ID NO: 10 and the amino acid sequence shown in SEQ ID NO: 2; (3) the amino acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 13; (4) the amino acid sequence shown in SEQ ID NO: 10 and the amino acid sequence shown in SEQ ID NO: 13; (5) the amino acid sequence shown in SEQ ID NO: 14 and the amino acid sequence shown in SEQ ID NO: 15; (6) the amino acid sequence shown in SEQ ID NO: 19 and the amino acid sequence shown in SEQ ID NO: 15; (7) The amino acid sequence shown in SEQ ID NO: 14 and the amino acid sequence shown in SEQ ID NO: 16; (8) The amino acid sequence shown in SEQ ID NO: 19 and the amino acid sequence shown in SEQ ID NO: 16; (9) The amino acid sequence shown in SEQ ID NO: 39 and the amino acid sequence shown in SEQ ID NO: 2; (10) The amino acid sequence shown in SEQ ID NO: 40 and the amino acid sequence shown in SEQ ID NO: 41; (11) The amino acid sequence shown in SEQ ID NO: 44 and the amino acid sequence shown in SEQ ID NO: 13; (12) The amino acid sequence shown in SEQ ID NO: 45 and the amino acid sequence shown in SEQ ID NO: 15; (13) The amino acid sequence shown in SEQ ID NO: 40 and the amino acid sequence shown in SEQ ID NO: 15, or (14) The amino acid sequence shown in SEQ ID NO: 45 and the amino acid sequence shown in SEQ ID NO: 41; The antibody or antigen-binding fragment thereof according to any one of claims 4 to 6, comprising a heavy chain variable region and a light chain variable region comprising:

8. wherein at least two Fab arms of said antibody or antigen-binding fragment thereof are identical or different; wherein the at least two Fab arms are identical and comprise a combination of identical amino acid sequences selected from any one of (1) to (11) as defined in claim 6, or a combination of identical amino acid sequences selected from any one of (1) to (14) as defined in claim 7. The antibody or antigen-binding fragment thereof according to any one of claims 4 to 7.

9. The at least two Fab arms are different and comprise a combination of different amino acid sequences selected from any one of (1) to (11) defined in claim 6, or a combination of different amino acid sequences selected from any one of (1) to (14) defined in claim 7; Preferably, at least two Fab arms of the antibody or antigen-binding fragment thereof comprise the same combination of light chain CDR sequences or comprise the same light chain variable region sequences. The antibody or antigen-binding fragment thereof according to any one of claims 4 to 8.

10. The antibody or antigen-binding fragment thereof (1) In one Fab arm, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 28, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 30, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 33, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 32; and In the other Fab arm, it comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:34, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:24, and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:31, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:32, or (2) In one Fab arm, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32; and In the other Fab arm, it comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:34, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:24, and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:32, or (3) In one Fab arm: HCDR1 having the amino acid sequence shown in SEQ ID NO: 54, HCDR2 having the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 56, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 31, LCDR2 having the amino acid sequence shown in SEQ ID NO: 33, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 32; and In the other Fab arm, it comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:48, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:51, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:50, and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:31, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:32, or (4) In one Fab arm: HCDR1 having the amino acid sequence shown in SEQ ID NO: 54, HCDR2 having the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 56, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 52, LCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 53; and The antibody or antigen-binding fragment thereof according to any one of claims 4 to 9, characterized in that the other Fab arm comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:48, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:51, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:50, and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:52, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

53.

11. The antibody or antigen-binding fragment thereof (1) In one Fab arm, a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15, and In the other Fab arm: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, or (2) In one Fab arm, a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16, and In the other Fab arm: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16, or (3) In one Fab arm: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15; and In the other Fab arm: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15; or (4) In one Fab arm: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41; and The antibody or antigen-binding fragment thereof according to any one of claims 4 to 10, characterized in that it comprises, in the other Fab arm: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

41.

12. Use of a polypeptide as defined in any one of claims 1 to 3 in the construction of an antibody.

13. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide as defined in any one of claims 1 to 3.

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