An antibody binding egfr and / or b7-h3 and uses thereof

Abstract

The present application provides a polypeptide as CDRs in an antibody light chain, light chain variable region or light chain, which is capable of constructing an antibody with CDRs in an antibody heavy chain, heavy chain variable region or heavy chain having binding affinity and / or specificity to different target proteins (antigens), the antibody retaining binding affinity and / or specificity to the target proteins (antigens) and biological activity. The present application also provides an anti-B7-H3 and / or EGFR antibody comprising the polypeptide as a light chain variable region or light chain.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims priority to Chinese invention patent application No. CN202411671052.1, filed on November 21, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention relates to the field of biomedicine, and more specifically, to a peptide and its use as an antibody light chain, and to antibodies comprising the peptide as a common light chain that bind EGFR and / or B7H3. Background Technology

[0004] Epidermal growth factor receptor (EGFR) is a transmembrane receptor that, during normal physiological processes, initiates signaling pathways for cell proliferation, differentiation, and survival by binding to epidermal growth factor (EGF). However, EGFR is overexpressed and activated in various tumors, often associated with tumor development, progression, invasiveness, and drug resistance. Studies have found that EGFR activation promotes tumor cell proliferation, survival, migration, and metastasis through a series of downstream signaling pathways (such as RAS / MAPK and PI3K / AKT). Therefore, EGFR is considered an important anti-cancer target, particularly in colorectal cancer, non-small cell lung cancer, head and neck cancer, and other cancer types. Common EGFR inhibitors include monoclonal antibodies (such as Cetuximab) and small molecule tyrosine kinase inhibitors (such as Gefitinib and Erlotinib).

[0005] B7-H3 (also known as CD276) is a member of the immune checkpoint molecule family and typically plays a role in the regulation of the immune system. As part of the B7 family, B7-H3 participates in the regulation of immune responses by binding to receptors on T cells. Under normal circumstances, the primary function of B7-H3 is to suppress T cell activation to prevent an overactive immune response. However, in many types of tumors, B7-H3 expression is often upregulated and associated with tumor immune escape. By inhibiting the immune system's recognition and attack on tumors, B7-H3 helps tumor cells evade immune surveillance, thereby promoting tumor growth and metastasis. Therefore, B7-H3 is considered a potential target for tumor immunotherapy.

[0006] In certain tumor types, EGFR and B7-H3 may co-involve in tumor immune evasion and tumor growth promotion. For example, through bioinformatics analysis, the inventors found that B7-H3 expression is associated with EGFR expression in lung cancer, head and neck cancer, pancreatic cancer, and esophageal cancer. Co-expression of B7-H3 and EGFR can enhance the inhibitory effect of tumor cells on the immune system and may enhance tumor invasiveness. This makes simultaneous targeting of EGFR and B7-H3 a potentially effective therapeutic strategy, especially in cases of severe tumor immune evasion. Furthermore, simultaneous targeting of EGFR and B7-H3, by achieving dual inhibition of tumors, is expected to improve efficacy while reducing the side effects of single-target therapy.

[0007] Bispecific antibodies are a common type of artificial antibody in this field, possessing two specific antigen-binding sites. Current conventional methods for preparing 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 are assembled into an antibody, light and heavy chain mismatches may occur. Therefore, it has been proposed to provide a polypeptide that can act as a light chain to pair with any heavy chain that has affinity and binding specificity to a specific target protein (such as EGFR or B7-H3 as proposed in this paper), thereby constructing an antibody (homodimer). Furthermore, this polypeptide can also act as a universal light chain to pair with two or more heavy chains that have binding affinity and specificity to different target proteins, thereby constructing bispecific or even multispecific antibodies (heterodimers, heteromultimers) with binding affinity and specificity to two or more target proteins (such as EGFR and B7-H3 as proposed in this paper). Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a polypeptide, in which the light chain, light chain variable region, or CDRs in the light chain of an antibody can be used to construct an antibody with an antibody heavy chain, heavy chain variable region, or CDRs in the heavy chain that have binding affinity and / or specificity to a certain target protein (antigen), or with two or more antibody heavy chains, heavy chain variable regions, or CDRs in the heavy chain that have binding affinity and / or specificity to different target proteins (antigens). Another object of the present invention is to provide an antibody comprising the said polypeptide as a light chain, light chain variable region, or CDRs in the light chain, for example, in the form of a homodimer or heterodimer.

[0009] The technical solution of the present invention is as follows.

[0010] In a first aspect, the present invention provides a polypeptide, the amino acid sequence of which comprises:

[0011] (i) the amino acid sequence shown in SEQ ID NO. 31; the amino acid sequence shown in SEQ ID NO. 32; and the amino acid sequence shown in SEQ ID NO. 33; or

[0012] (ii) The amino acid sequence shown in SEQ ID NO.31; the amino acid sequence shown in SEQ ID NO.32; and the amino acid sequence shown in SEQ ID NO.41.

[0013] SEQ ID NO.31: SASSVSSVH

[0014] SEQ ID NO.32: GTSKLAS

[0015] SEQ ID NO.33: QQWSGNPPYT

[0016] SEQ ID NO.41: QLWSGNPPYT

[0017] Further, the polypeptide comprises the amino acid sequence shown in SEQ ID NO.11, SEQ ID NO.14 or SEQ ID NO.40, or comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.11, SEQ ID NO.14 or SEQ ID NO.40.

[0018] SEQ ID NO.11:

[0019] RIVLTQSPGFMSASPGEKVTMTCSASSSVSSVHWFQQKSGTSPKRWIYGTSKLASG VPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSGNPPYTFGGGTKLEIKRA

[0020] SEQ ID NO.14:

[0021] DIQLTQSPSSSLSASVGDRVTITCSASSSVSSVHWFQQKPGTSPKRWIYGTSKLASGVP SRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSGNPPYTFGQGTRLEIK

[0022] SEQ ID NO.40:

[0023] DIQLTQSPSSSLSASVGDRVTITCSASSSVSSVHWFQQKPGTSPKRWIYGTSKLASGV PSRFSGSGSGTDYTLTISSLQPEDFATYYCQLWSGNPPYTFGQGTRLEIK

[0024] Further, the polypeptide comprises the amino acid sequence shown in SEQ ID NO.16, SEQ ID NO.25 or SEQ ID NO.39, or comprises an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.16, SEQ ID NO.25 or SEQ ID NO.39.

[0025] SEQ ID NO.16:

[0026] RIVLTQSPGFMSASPGEKVTMTCSASSSVSSVHWFQQKSGTSPKRWIYGTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSGNPPYTFGGGTKLEIKR ARTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0027] SEQ ID NO.25:

[0028] DIQLTQSPSSSLSASVGDRVTITCSASSSVSSVHWFQQKPGTSPKRWIYGTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSGNPPYTFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0029] SEQ ID NO.39:

[0030] DIQLTQSPSSSLSASVGDRVTITCSASSSVSSVHWFQQKPGTSPKRWIYGTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQLWSGNPPYTFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0031] In the context of this invention, the term "at least 75% identity" encompasses any percentage of identity between two amino acid sequences, ranging from at least 75% to 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0032] The aforementioned polypeptides are derived from specific polypeptides or antibodies provided in the embodiments of this application. Experiments have demonstrated that the polypeptides provided by this invention, as antibody light chains, light chain variable regions, or antigen complementarity-determining regions (CDRs) in light chains, can respectively bind to the heavy chains, heavy chain variable regions, or heavy chain CDRs of antibodies that have binding affinity and / or specificity to different target proteins (antigens) to construct antibodies. The antibodies retain or have stronger binding affinity and / or specificity to the target protein (antigen) and the corresponding biological activity.

[0033] The constructed antibody can have any structural form, such as a monoclonal antibody or antibody forms like scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv. For example, the polypeptide, as the light chain, light chain variable region, or CDRs in the light chain of an antibody, can be combined with the heavy chain, heavy chain variable region, or CDRs in the heavy chain of an antibody that has binding affinity and / or specificity to a target protein (antigen) to construct an antibody that still has binding affinity and / or specificity to that target protein (antigen). This antibody is a homodimeric antibody (a monospecific antibody) having two identical heavy chains and two identical light chains. Alternatively, for example, the polypeptide, as the light chain, light chain variable region, or CDRs in the light chain of an antibody, can be combined with the heavy chain, heavy chain variable region, or CDRs in the heavy chain of two antibodies that have binding affinity and / or specificity for different target proteins (antigens) to construct an antibody that still has binding affinity and / or specificity for that target protein (antigen). This antibody is a heterodimeric antibody (a bispecific antibody) having two different heavy chains and two identical light chains. Furthermore, the polypeptide provided by this invention can also be used to construct multispecific antibodies with binding affinity and / or specificity for a wider range of target proteins (antigens).

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

[0035] Thirdly, the present invention provides the use of the polypeptide described in the first aspect or the nucleic acid molecule described in the second aspect in the construction of antibodies.

[0036] Experiments have shown that, as an antibody light chain or light chain variable region, the polypeptide provided in the first aspect of the present invention can be used with the heavy chain or antibody heavy chain variable region of an antibody that has binding affinity and / or specificity to different target proteins (antigens) to construct new antibodies, wherein the antibodies retain (or even have stronger) binding affinity and / or specificity to the target protein (antigen) and biological activity, etc.

[0037] According to a specific embodiment of the present invention, the target protein may be epidermal growth factor receptor (EGFR) or B7 family protein B7-H3. In the context of the present invention, EGFR or B7-H3 may be mammalian, such as primate or rodent EGFR or B7-H3, more preferably human EGFR or B7-H3.

[0038] Accordingly, as described in the first aspect above, the constructed antibody can 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 can serve as the light chain or a variable region of the light chain of the antibody. For example, the constructed antibody is a bispecific antibody, and the polypeptide provided in the first aspect of the present invention serves as the common light chain of the bispecific antibody; the bispecific antibody is such as the EGFR×B7-H3 bispecific antibody provided by the present invention.

[0039] Fourthly, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a polypeptide provided in the first aspect of the present invention as an antibody light chain, a light chain variable region, or CDRs in the light chain.

[0040] In the context of this invention, the antigen-binding fragment is any functional fragment of an antibody capable of specifically binding to a target protein. In the context of this invention, "target protein" and "target" and "antigen" are used interchangeably.

[0041] The antibodies or antigen-binding fragments provided by this invention can be IgG-like antibodies, or antibody forms such as scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv. In this invention, "IgG-like antibody" refers to an antibody or antigen-binding fragment having two Fab arms and an optional Fc region, and is a traditional monoclonal antibody (mAb) structure or a similar structure. In this invention, "Fab arm" refers to a Fab fragment (Antigen-binding fragment), which consists of a complete light chain (variable and constant regions) and a partial heavy chain structure (variable region and a constant region fragment), with the light and heavy chains linked by disulfide bonds. Regarding target protein (antigen) binding properties, the antibodies or antigen-binding fragments provided by this invention can be bispecific antibodies.

[0042] Preferably, the antibody or antigen-binding fragment provided by the present invention may contain the amino acid sequence provided by the first aspect of the present invention as light chain CDRs in its light chain variable region; or contain the amino acid sequence shown in SEQ ID NO.11, SEQ ID NO.14 or SEQ ID NO.40, or contain an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.11, SEQ ID NO.14 or SEQ ID NO.40 as a light chain variable region (VL). Alternatively, the antibody or antigen-binding fragment provided by the present invention may contain the amino acid sequence shown in SEQ ID NO.16, SEQ ID NO.25 or SEQ ID NO.39, or contain an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.16, SEQ ID NO.25 or SEQ ID NO.39 as a light chain (LC).

[0043] Furthermore, the antibody or its antigen-binding fragment provided by the present invention may further include a heavy chain variable region (VH), wherein the heavy chain variable region forms a binding domain for the target protein with the antibody light chain, the light chain variable region, or CDRs in the light chain. According to a specific embodiment of the present invention, the target protein may be B7-H3 or EGFR.

[0044] According to a specific embodiment of the present invention, the antibody or its antigen-binding fragment respectively comprises heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and light chain CDRs (LCDR1, LCDR2 and LCDR3) as shown below:

[0045] (1) HCDR1 containing the amino acid sequence shown in SEQ ID NO.28, HCDR2 containing the amino acid sequence shown in SEQ ID NO.29, HCDR3 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.33;

[0046] (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO.34, HCDR2 containing the amino acid sequence shown in SEQ ID NO.35, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.33;

[0047] (3) HCDR1 containing the amino acid sequence shown in SEQ ID NO.28, HCDR2 containing the amino acid sequence shown in SEQ ID NO.29, HCDR3 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.41;

[0048] (4) HCDR1 containing the amino acid sequence shown in SEQ ID NO.34, HCDR2 containing the amino acid sequence shown in SEQ ID NO.38, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.41.

[0049] Furthermore, the antibody or its antigen-binding fragment may comprise a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDRs and the framework regions (FRs) therebetween, and the arrangement of the regions may be FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further, in terms of the amino acid sequence, the heavy chain variable region and the light chain variable region respectively comprise:

[0050] (1) The amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11;

[0051] (2) The amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 14;

[0052] (3) The amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11;

[0053] (4) The amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 14;

[0054] (5) The amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO. 40 or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0055] (6) The amino acid sequence shown in SEQ ID NO.37 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.40 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.40.

[0056] The antibody or its antigen-binding fragment may be a mouse antibody, a chimeric antibody, or a humanized antibody; and may further comprise a heavy chain constant region and / or a light chain constant region. According to a specific embodiment of the present invention, the heavy chain constant region may be the heavy chain constant region of IgG1 or IgG4; the light chain constant region may be a kappa or lambda light chain constant region. For example, the heavy chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO. 17 (hIgG1) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO. 18 (hkappa) or an amino acid sequence having at least 75% identity with the amino acid sequence. Alternatively, the heavy chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.19 (hIgG1; Knob) or SEQ ID NO.20 (hIgG1; Hole) or an amino acid sequence having at least 75% identity with said amino acid sequence; the light chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.18 or an amino acid sequence having at least 75% identity with said amino acid sequence. Alternatively, the heavy chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.21 (hIgG1-LALA; Knob) or SEQ ID NO.22 (hIgG1-LALA; Hole) or an amino acid sequence having at least 75% identity with said amino acid sequence; the light chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.18 or an amino acid sequence having at least 75% identity with said amino acid sequence.

[0057] The antibody or antigen-binding fragment provided by the present invention may have at least two VH+VL domain combinations (further, at least two Fab arms), said domain combinations may be identical to each other, thereby binding to the same target protein (antigen). In this case, the antibody or antigen-binding fragment contains the same amino acid sequence combination selected from HCDR1 to HCDR3 and LCDR1 to LCDR3 provided above in its at least two VH+VL domain combinations, or contains the same amino acid sequence combination selected from the heavy chain variable region and light chain variable region provided above in (1) to (6). The antibody or antigen-binding fragment also contains a heavy chain constant region and a light chain constant region. For example, as described above, the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO. 17; the light chain constant region contains the amino acid sequence shown in SEQ ID NO. 18. Further, the antibody or antigen-binding fragment has a heavy chain and a light chain, for example, a monoclonal antibody having the same two heavy chains and the same two light chains.

[0058] Alternatively, the at least two VH+VL domain combinations (further, at least two Fab arms) of the antibody or its antigen-binding fragment provided by the present invention may be different from each other, thereby binding to different target proteins (antigens). In this case, the antibody contains different amino acid sequence combinations selected from HCDR1 to HCDR3 and LCDR1 to LCDR3 provided above (1) to (4), or different amino acid sequence combinations selected from the heavy chain variable region and light chain variable region provided above (1) to (6). In this case, it is preferred that the at least two VH+VL domain combinations (further, at least two Fab arms) of the antibody or its antigen-binding fragment contain the same light chain CDRs (LCDR1 to LCDR3) sequence combination, or contain the same light chain VL sequence. For example, each of the at least two VH+VL domain combinations (further, at least two Fab arms) comprises: LCDR1 containing the amino acid sequence shown in SEQ ID NO. 31, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO. 33; or LCDR1 containing the amino acid sequence shown in SEQ ID NO. 31, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO. 41. Further, each of the two VH+VL domain combinations (further, two Fab arms) comprises: a light chain variable region containing the amino acid sequence shown in SEQ ID NO. 11, SEQ ID NO. 14, or SEQ ID NO. 40, or a light chain variable region containing an amino acid sequence having at least 75% identity with the amino acid sequence.

[0059] According to a specific embodiment of the present invention, the antibody or its antigen-binding fragment is as follows:

[0060] (1) The first VH+VL domain combination of B7-H3 (further, Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO.28, HCDR2 containing the amino acid sequence shown in SEQ ID NO.29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing SEQ ID NO.33;

[0061] The second VH+VL domain combination of EGFR (further, the Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 34, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 35, and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 31, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 32, and LCDR3 containing SEQ ID NO. 33; or

[0062] (1) The first VH+VL domain combination of B7-H3 (further, Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO.28, HCDR2 containing the amino acid sequence shown in SEQ ID NO.29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing SEQ ID NO.41;

[0063] The second VH+VL domain combination of EGFR (further, the Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 34, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 38, and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 31, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 32, and LCDR3 containing SEQ ID NO. 41.

[0064] According to a specific embodiment of the present invention, the antibody or its antigen-binding fragment is further described as follows:

[0065] (1) The first VH+VL domain combination (further, the Fab arm) of B7-H3, wherein the first VH+VL domain combination comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 75% identity with said amino acid sequence; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity with said amino acid sequence; and

[0066] The second VH+VL domain combination of EGFR (further, the Fab arm) comprises: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; and a light chain variable region containing the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0067] (2) The first VH+VL domain combination of B7-H3 (further, the Fab arm), wherein the first VH+VL domain combination comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 75% identity with said amino acid sequence; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 40 or an amino acid sequence having at least 75% identity with said amino acid sequence; and

[0068] The second VH+VL domain combination of EGFR (further, the Fab arm) comprises: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO. 37 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 40 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 40.

[0069] In this case, the antibody or its antigen-binding fragment may further comprise a heavy chain constant region and / or a light chain constant region. According to a specific embodiment of the invention, the heavy chain constant region may be the heavy chain constant region of IgG1 or IgG4; the light chain constant region may be the kappa or lambda light chain constant region. For example, the heavy chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO. 17 (hIgG1) or an amino acid sequence having at least 75% identity with said amino acid sequence; the light chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO. 18 (hkappa) or an amino acid sequence having at least 75% identity with said amino acid sequence. Alternatively, the heavy chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO. 19 (hIgG1; Knob) or SEQ ID NO. 20 (hIgG1; Hole) or an amino acid sequence having at least 75% identity with said amino acid sequence; the light chain constant region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 75% identity with said amino acid sequence. Alternatively, the heavy chain constant region of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO.21 (hIgG1-LALA; Knob) or SEQ ID NO.22 (hIgG1-LALA; Hole) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO.18 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0070] Furthermore, the antibody is a bispecific antibody, which has two different heavy chains but contains the same two light chains. According to a specific embodiment of the present invention, the bispecific antibody may contain the amino acid sequence shown in SEQ ID NO. 25 as the light chain.

[0071] According to specific embodiments of the present invention, the bispecific antibodies provided by the present invention are shown in Tables 11 and 19 in the "Specific Embodiments" section. The bispecific antibodies in Tables 11 and 19 include the following structural domains:

[0072] The bispecific antibody H125C3Y3CV2xH31A2-4V2-hIgG1 comprises two Fab arms targeting B7-H3 and EGFR, respectively. One Fab arm contains a heavy chain variable region (SEQ ID NO.13) and a light chain variable region (SEQ ID NO.14) targeting B7-H3, and the other Fab arm contains a heavy chain variable region (SEQ ID NO.15) and a light chain variable region (SEQ ID NO.14) targeting EGFR. This bispecific antibody contains two identical light chain variable regions (SEQ ID NO.14).

[0073] The bispecific antibody H125C3Y3CV2xH31A2Y4CV8-CLC129-hIgG1m39x40 comprises two Fab arms targeting B7-H3 and EGFR, respectively. One Fab arm contains a heavy chain variable region (SEQ ID NO.13) and a light chain variable region (SEQ ID NO.40) targeting B7-H3, and the other Fab arm contains a heavy chain variable region (SEQ ID NO.37) and a light chain variable region (SEQ ID NO.40) targeting EGFR. The bispecific antibody contains two identical light chain variable regions (SEQ ID NO.40).

[0074] Furthermore, the bispecific antibodies in Tables 11 and 19, each being an IgG type antibody, contain two different heavy chains (heavy chain 1 and heavy chain 2) and the same two light chains:

[0075] The bispecific antibody H125C3Y3CV2xH31A2-4V2-hIgG1 contains the amino acid sequence shown in SEQ ID NO.23 as heavy chain 1, the amino acid sequence shown in SEQ ID NO.24 as heavy chain 2, and the amino acid sequence shown in SEQ ID NO.25 as the same light chain.

[0076] The bispecific antibody H125C3Y3CV2xH31A2Y4CV8-CLC129-hIgG1m39x40 contains the amino acid sequence shown in SEQ ID NO.26 as heavy chain 1, the amino acid sequence shown in SEQ ID NO.27 as heavy chain 2, and the amino acid sequence shown in SEQ ID NO.39 as the same light chain.

[0077] Fifthly, the present invention provides an antibody-drug conjugate comprising the polypeptide described in the first aspect of the present invention or the antibody described in the fourth aspect of the present invention or its antigen-binding fragment, wherein the polypeptide or the antibody or its antigen-binding fragment is conjugated to a small molecule toxic compound via a linker.

[0078] In the antibody-drug conjugates provided by this invention, the small molecule toxic compound may be a microtubule inhibitor (such as MMAE), a DNA cross-linking agent (such as PBD dimer), a topoisomerase 1 inhibitor (such as Exatecan), etc.

[0079] In the antibody-drug conjugates provided by this invention, the linker can be a cleavable linker, such as a disulfide linker, a Val-Cit linker, etc.

[0080] In a sixth aspect, the present invention provides a composition comprising the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the antibody described in the fourth aspect, or an antigen-binding fragment thereof, or the antibody-drug conjugate described in the fifth aspect. The composition is preferably a pharmaceutical composition, which may further optionally contain pharmaceutically acceptable excipients, carriers, or excipients. The pharmaceutical composition can be formulated into various dosage forms known in the medical or pharmaceutical fields and administered in an applicable manner.

[0081] In a seventh aspect, the present invention provides the use of the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the antibody described in the fourth aspect or its antigen-binding fragment, the pharmaceutical composition described in the fifth aspect, or the antibody-drug conjugate described in the sixth aspect in the preparation of a medicament for treating or alleviating a disease.

[0082] The disease can be a tumor or cancer, such as a tumor or cancer expressing B7-H3 and / or EGFR. For example, the tumor or cancer has elevated expression of B7-H3 and / or EGFR. Preferably, the tumor or cancer moderately or highly expresses B7-H3 and / or EGFR. According to specific embodiments of the invention, the disease can be, for example, small cell lung cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, head and neck squamous cell carcinoma, esophageal cancer, biliary tract cancer, melanoma, endometrial cancer, ovarian cancer, breast cancer, cervical cancer, osteosarcoma, gastric adenocarcinoma, pancreatic cancer, colorectal cancer, or liver cancer.

[0083] Eighthly, the present invention provides a method for treating or alleviating a disease, the method comprising administering to a subject in need the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the antibody described in the fourth aspect or an antigen-binding fragment thereof, the pharmaceutical composition described in the fifth aspect, or the antibody-drug conjugate described in the sixth aspect of the present invention.

[0084] The method provided by this invention is used to treat or alleviate tumors or cancers, such as tumors or cancers expressing B7-H3 and / or EGFR. For example, the tumor or cancer has elevated expression of B7-H3 and / or EGFR. Preferably, the tumor or cancer moderately or highly expresses B7-H3 and / or EGFR. According to specific embodiments of the invention, the disease can be, for example, small cell lung cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, head and neck squamous cell carcinoma, esophageal cancer, biliary tract cancer, melanoma, endometrial cancer, ovarian cancer, breast cancer, cervical cancer, osteosarcoma, gastric adenocarcinoma, pancreatic cancer, colorectal cancer, or liver cancer.

[0085] The subjects are mammals, preferably primates or rodents, and more preferably humans.

[0086] In a ninth aspect, the present invention also provides an antibody or antigen-binding fragment thereof that binds to B7-H3, said antibody or antigen-binding fragment comprising heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and light chain CDRs (LCDR1, LCDR2 and LCDR3) as shown below:

[0087] The HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 28, the HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 29, and the HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 30; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 31, the LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 32, and the LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 33.

[0088] Preferably, the antibody binding to B7-H3 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise:

[0089] (1) The amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0090] (2) The amino acid sequence shown in SEQ ID NO.13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.14.

[0091] The antibody or antigen-binding fragment of B7-H33 provided by this invention can be an IgG-like antibody, or an antibody in the form of scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

[0092] The antibody or its antigen-binding fragment may be a murine antibody, a chimeric antibody, or a humanized antibody; and may further comprise a heavy chain constant region and a light chain constant region. According to a specific embodiment of the present invention, the heavy chain constant region may be the heavy chain constant region of IgG1 or IgG4; the light chain constant region may be the kappa or lambda light chain constant region. For example, the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.17 (hIgG1) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.18 (hkappa) or an amino acid sequence having at least 75% identity with the amino acid sequence. Alternatively, the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.19 (hIgG1; Knob) or SEQ ID NO.20 (hIgG1; Hole) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.18 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0093] In a tenth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof that binds to B7-H3 as described in the ninth aspect in the preparation of a medicament for treating or alleviating a disease. The medicament may be an antibody-based drug used to treat or alleviate diseases related to the expression of its target protein or antigen, such as diseases related to B7-H3 expression.

[0094] The disease can be a tumor or cancer, such as a tumor or cancer expressing B7-H3. For example, the tumor or cancer has elevated expression of B7-H3. Preferably, the tumor or cancer moderately or highly expresses B7-H3. According to specific embodiments of the invention, the disease can be, for example, small cell lung cancer, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, head and neck squamous cell carcinoma, esophageal cancer, biliary tract cancer, melanoma, endometrial cancer, ovarian cancer, breast cancer, cervical cancer, or osteosarcoma.

[0095] Correspondingly, the present invention also provides a method for treating or alleviating the disease, the method comprising administering to a subject requiring treatment an antibody or antigen-binding fragment thereof that binds to B7-H3 as described herein. The subject is a mammal, preferably a primate or rodent, more preferably a human.

[0096] Eleventhly, the present invention also provides an antibody or antigen-binding fragment thereof that binds to EGFR, said antibody or antigen-binding fragment comprising heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and light chain CDRs (LCDR1, LCDR2 and LCDR3) as shown below:

[0097] The HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 34, the HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 35, and the HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 36; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, the LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and the LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31.

[0098] Preferably, the antibody binding to EGFR or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise:

[0099] (1) The amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0100] (2) The amino acid sequence shown in SEQ ID NO.15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.14.

[0101] The antibody or antigen-binding fragment of PD-L1 provided by this invention can be an IgG-like antibody, or an antibody in the form of scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

[0102] The antibody or its antigen-binding fragment may be a murine antibody, a chimeric antibody, or a humanized antibody; and may further comprise a heavy chain constant region and a light chain constant region. According to a specific embodiment of the present invention, the heavy chain constant region may be the heavy chain constant region of IgG1 or IgG4; the light chain constant region may be the kappa or lambda light chain constant region. For example, the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.17 (hIgG1) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.18 (hkappa) or an amino acid sequence having at least 75% identity with the amino acid sequence. Alternatively, the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.19 (hIgG1; Knob) or SEQ ID NO.20 (hIgG1; Hole) or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO.18 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0103] In a twelfth aspect, the present invention provides the use of the EGFR-binding antibody or its antigen-binding fragment as described in the eleventh aspect in the preparation of a medicament for treating or alleviating a disease. The medicament may be an antibody-based drug used to treat diseases related to the expression of its target protein or antigen, such as EGFR-related diseases.

[0104] The disease can be a tumor or cancer, such as a tumor or cancer expressing EGFR. For example, the tumor or cancer has elevated EGFR expression. Preferably, the tumor or cancer moderately or highly expresses EGFR. According to a specific embodiment of the invention, the disease can be, for example, non-small cell lung cancer, nasopharyngeal carcinoma, head and neck squamous cell carcinoma, gastric adenocarcinoma, pancreatic cancer, colorectal cancer, liver cancer, or breast cancer.

[0105] Correspondingly, the present invention also provides a method for treating or alleviating the disease, the method comprising administering to a subject requiring treatment an EGFR-binding antibody or antigen-binding fragment thereof as described in the present invention. The subject is a mammal, preferably a primate or rodent, more preferably a human.

[0106] This invention provides a polypeptide that, experimentally demonstrated, serves as an antibody light chain, light chain variable region, or CDR in the light chain. This polypeptide can be used to construct antibodies by binding with antibody heavy chains, heavy chain variable regions, or heavy chain CDRs that have binding affinity and / or specificity to different target proteins (antigens). The antibodies retain or even exhibit higher binding affinity and / or specificity to the target proteins (antigens) and corresponding biological activity. Therefore, the polypeptide provided by this invention has the potential to be used as a common light chain variable region or light chain for constructing bispecific or more specific antibodies or other antibody-based drug molecules (e.g., antibody-drug conjugates). Furthermore, this invention also provides antibodies comprising the polypeptide as a common light chain variable region or light chain, such as anti-B7-H3 antibodies, anti-EGFR antibodies, anti-PD-L1 antibodies, and B7-H3×EGFRPDL1 bispecific antibodies, as well as antibody-drug conjugates conjugated with any of these antibodies. Attached Figure Description

[0107] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0108] Figure 1 The binding activity of the anti-B7-H3 murine antibody to human or monkey B7-H3 overexpressing cells was demonstrated;

[0109] Figure 2 The binding activity of the anti-B7-H3 humanized antibody to human B7-H3 overexpressing cells or human tumor cells was demonstrated.

[0110] Figure 3 The binding activity of the anti-B7-H3 humanized antibody to monkey B7-H3 protein was demonstrated;

[0111] Figure 4 The internalization activity of the anti-B7-H3 humanized antibody on tumor cells was demonstrated;

[0112] Figure 5 The binding activity of anti-B7-H3 or anti-EGFR humanized antibodies, B7-H3×EGFR bispecific antibodies to human EGFR, human B7-H3 overexpressing cells and human tumor cells was demonstrated.

[0113] Figure 6 The binding activity of anti-B7-H3 or anti-EGFR humanized antibodies, B7-H3×EGFR bispecific antibodies to human EGFR, human B7-H3 overexpressing cells and human tumor cells was demonstrated.

[0114] Figure 7 The blocking activity of anti-EGFR humanized antibody and B7-H3×EGFR bispecific antibody against EGFR-EGF binding was demonstrated.

[0115] Figures 8 to 11The binding activity of the B7-H3×EGFR bispecific antibody against EGFR, B7-H3 family proteins or EGFR, B7-H3 proteins from different species was demonstrated.

[0116] Figure 12 The internalization activity of the B7-H3×EGFR bispecific antibody on EGFR and / or B7-H3 expressing tumor cells was demonstrated.

[0117] Figure 13 The binding activity of B7-H3×EGFR ADC to EGFR and / or B7-H3 expressing cells was demonstrated;

[0118] Figure 14 The internalization activity of B7-H3×EGFR ADC in EGFR and / or B7-H3 expressing cells was demonstrated;

[0119] Figure 15 The B7-H3×EGFR ADC was shown to have cytotoxic activity against EGFR and / or B7-H3-expressing human tumor cells;

[0120] Figure 16 The bystander effect of B7-H3×EGFR ADC is shown;

[0121] Figures 17 to 19 The curves showing the changes in tumor volume and body weight over time in mice after drug administration are presented in different mouse models of tumors.

[0122] Figure 20 The physicochemical properties analysis results of the modified B7-H3×EGFR bispecific antibody are shown;

[0123] Figure 21 The binding activity of the B7-H3×EGFR bispecific antibody before and after modification to human EGFR and B7-H3 proteins was demonstrated.

[0124] Figure 22 The binding activity of the B7-H3×EGFR bispecific antibody before and after modification with EGFR and / or B7-H3 overexpressing cells was demonstrated.

[0125] Figure 23 The internalization activity of the B7-H3×EGFR bispecific antibody before and after modification on human tumor cells was demonstrated.

[0126] The best way to implement an invention

[0127] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0128] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0129] The following constant region sequence was used when constructing the antibody:

[0130] Human heavy chain CH1 and FC sequences (SEQ ID NO.17):

[0131] hIgG1-WT:

[0132] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0133] GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0134] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0135] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0136] RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

[0137] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0138] Human light chain CL sequence (SEQ ID NO.18):

[0139] hkappa:

[0140] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

[0141] DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0142] Human heavy chain Knob CH1 and FC sequences (FC: hIgG1; knob: S354C / T366W; SEQ ID NO.19):

[0143] Human heavy chain Hole CH1 and FC sequences (FC: hIgG1; hole: Y349C / T366S / L368A / Y407V; SEQ ID NO. 20):

[0144]

[0145] Human heavy chain Knob CH1 and FC sequences - LALA (FC: hIgG1, L234A / L235A; knob: S354C / T366W; SEQ ID NO.21):

[0146]

[0147] Human heavy chain Hole CH1 and FC sequences - LALA (FC: hIgG1, L234A / L235A; hole: Y349C / T366S / L368A / Y407V; SEQ ID NO.22):

[0148]

[0149] Example 1 Screening of common light chains

[0150] Analysis of existing technologies revealed that KV4-type germlines are expressed at a relatively high frequency in mice and pair with mouse heavy chains at a higher frequency. Therefore, this type of germline sequence was selected, and the following mouse germline sequence KV4-59 was chosen from it because it is relatively easy to humanize, has good solubility, low immunogenicity, and no potential modification sites.

[0151] KV4-59 (SEQ ID NO.1):

[0152] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPA

[0153] RFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPP FGSGTKLEIK

[0154] The above sequence was modified. Since CDR3 of the light chain (see underlined portion; obtained according to the KABAT definition) plays a crucial role in the affinity between the antibody and antigen, an amino acid was added after CDR3 to enhance the affinity for potential antigens. The following three sequences were obtained:

[0155] KV4-59-1 (SEQ ID NO.2):

[0156]

[0157] KV4-59-2 (SEQ ID NO.3):

[0158]

[0159] KV4-59-3 (SEQ ID NO.4):

[0160]

[0161] The above three sequences were used as the variable region sequences of the antibody light chain, and chimeric antibodies were constructed by combining them with the variable region sequences of the heavy chain of the mouse anti-chicken egg lysozyme antibody and the constant regions of the human heavy and light chains.

[0162] The heavy chain variable region sequence of mouse anti-chicken egg lysozyme antibody (SEQ ID NO.5):

[0163] LDVKLQESGPGLVKPSQSLSLTCSVTGYSFTSGYYWNWIRLFPGNKLEWMGYVSYFGT

[0164] NNYNPSLKNRFSITRDTSKNQFFLKLNSVSTEDTATYYCARGEEWDWSFDVWGTGTTV

[0165] TVSS

[0166] By testing the antigen-binding affinity, expression level, thermal stability, solubility, and freeze-thaw stability of the three chimeric antibodies, it was confirmed that the antibody constructed with KV4-59-1 (SEQ ID NO.2) as the variable region sequence of the antibody light chain has better properties.

[0167] Example 2 Construction of transgenic mice

[0168] Based on the C57 mouse antibody lambda light chain gene sequence, sgRNAs were designed and validated in vitro using a Cas9-gRNA target efficiency assay kit (Vishanglide, Cat#: VK007-30T). Highly efficient sgRNA sequences were selected: TGAATGCCATGTACTTATGG (SEQ ID NO. 6) and AAGTTCAGCTCCTAAAATGG (SEQ ID NO. 7). These sgRNA sequences were synthesized and microinjected into mouse zygotes along with spCas91.1 protein. The sgRNA mediated Cas9 protein cleavage of the target gene fragment to be deleted, causing DNA double-strand breaks. DNA repair occurred at both ends of the genome via non-homologous end joining (NHEJ), while the target fragment in the middle was deleted, achieving gene knockout. Genotyping of mice two weeks after birth confirmed that the lambda gene had been knocked out.

[0169] Based on the kappa light chain sequence of the C57 mouse antibody, sgRNAs were designed and validated in vitro using a Cas9-gRNA target efficiency assay kit (Vishanglide, Cat#: VK007-30T). Highly efficient sgRNA sequences were selected: IGK-L1: GTGAATGCCATGTACTTATGG (SEQ ID NO.8); IGK-R1: CAAGTTCAGCTCCTAAAATGG (SEQ ID NO.9). These sgRNA sequences were synthesized and microinjected into mouse zygotes along with spCas91.1 protein. The sgRNA mediated the Cas9 protein to create a nick at the target DNA location. Using a Donor DNA fragment containing a homologous arm and the target fragment KV4-59-1 (SEQ ID NO.2), homologous recombination was used to knock the DNA fragment into the target location. Genotyping was performed on mice two weeks after birth, confirming the knock-in of the common light chain gene.

[0170] Heterozygous mice with lambda gene knockout were mated with heterozygous mice with common light chain knock-in. Genotyping was performed on each offspring 2 weeks after birth to detect common light chain gene knock-in and lambda knockout, until mice with all alleles of common light chain gene knock-in and both lambda and kappa knockout were obtained.

[0171] Example 3 Preparation of hybridoma cells secreting anti-B7-H3 antibodies

[0172] Mice constructed in Example 2 were immunized using B7H3-mFc (Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. (“Kangyuan Bochuang”), Cat#: KP-1112) as an immunogen. Mice with high titers were selected, and serum was collected. After dissection, the spleen was harvested, and spleen cells were isolated. These spleen cells were fused with cultured myeloma cells to obtain hybridoma cells. The binding activity of the hybridoma cell supernatant to the antigen protein B7H3-His (Kangyuan Bochuang, Cat#: KP-1114) was detected using ELISA, yielding multiple positive hybridoma cell lines secreting anti-B7-H3 antibodies.

[0173] A murine antibody against B7-H3 was obtained from a positive hybridoma cell line and named B7H3-KD-23-0449-125C3Y3C (abbreviated as "125C3Y3C" or "KD-23-0449"). Its variable region sequence is as follows (where the heavy chain and light chain CDRs are underlined and obtained according to the KABAT definition method, the same below):

[0174] B7H3-KD-23-0449-125C3Y3C (Mouse Antibody)

[0175] >VH (SEQ ID NO.10; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.28 / SEQ ID NO.29 / SEQ IDNO.30)

[0176] EVKLVESGGGLVKPGGSLKLSCAASGFTFS SYGMS WVRQSPEKRLEWVA TISGDSIFNY

[0177] YLDTVKG RFTISRDNVRNNLYLQMSSLRSEDTAVYYCAR QDYGSSWFAY WGQGTLVTV

[0178] SA

[0179] >VL(SEQ ID NO.11; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.31 / SEQ ID NO.32 / SEQ IDNO.33)

[0180] RIVLTQSPGFMSASPGEKVTMTC SASSSVSSVH WFQQKSGTSPKRWIY GTSKLAS GVPA

[0181] RFSGSGSGTSYSLTISSMEAEDAATYYC QQWSGNPPYT FGGGTKLEIKRA

[0182] Example 4 Detection of the binding activity of anti-B7-H3 murine antibody to human or monkey B7-H3 overexpressing cells

[0183] CT26-B7H3 cells (Kangyuan Bochuang, Cat#: KC-3675) overexpressing human B7-H3 and 293T-B7H3-KO-cyno-B7H3 cells (Kangyuan Bochuang, Cat#: KC-3228) overexpressing monkey B7-H3 and with endogenous human B7-H3 knocked out were collected in logarithmic growth phase. After washing with PBS, the cells were resuspended and cultured at 2×10⁻⁶. 4 Cells were seeded at a density of 50 μL / well in 96-well deep-well plates. The mouse antibody to be tested and the anti-B7-H3 control antibody Ifinatamab (Kangyuan Bochuang, Cat#: KB-1091) were serially diluted and incubated with the cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer (PBS + 2% FBS + 2 mMEDTA). Then, each well was incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09) or the anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307) at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer and resuspended. Flow cytometry was used to detect the antibody signal bound to the cells. Results are shown in the figure below. Figure 1 1A, 1B and Table 1.

[0184] Table 1. Results of detection of the binding activity of anti-B7-H3 murine antibody to human or monkey B7-H3 overexpressing cells.

[0185]

[0186]

[0187] Example 5 Detection of affinity of murine anti-B7-H3 antibody for human B7-H3 protein

[0188] The ForteBio Octet assay was used to detect the affinity between murine anti-B7-H3 antibody and human B7-H3 protein. Human B7H3-His (Kangyuan Bochuang, Cat#: KP-1114) was used as the mobile phase and diluted sequentially to 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM, with simultaneous detection. The results are shown in Table 2.

[0189] Table 2. Results of the detection of the affinity of anti-B7-H3 murine antibody for human B7-H3 protein

[0190] Antibody ka(1 / Ms) kd(1 / s) KD(M) Ifinatamab 5.20E+05 6.37E-05 1.22E-10 125C3Y3C 1.26E+06 4.24E-04 3.37E-10

[0191] Example 6 Construction of humanized and chimeric antibodies against B7-H3

[0192] (I) Construction of humanized antibodies

[0193] The heavy and light chain variable regions of the murine anti-B7H3 antibody were humanized to obtain a humanized antibody sequence. The coding sequences of the humanized antibody sequence were linked to the coding sequences of the heavy chain constant region (SEQ ID NO.17) of human IgG1 and the light chain constant region (SEQ ID NO.18) of human kappa, respectively. The resulting coding genes were cloned into a eukaryotic expression vector, expressed, and the humanized anti-B7H3 antibody was obtained, named B7H3-KA-2802-H125C3Y3CV2-hIgG1 (abbreviated as "H125C3Y3CV2-hIgG1" or "KA-2802"). Its heavy and light chain variable region sequences are shown below (where the heavy chain and light chain CDRs are underlined).

[0194] B7H3-KA-2802-H125C3Y3CV2-hIgG1 (humanized antibody)

[0195] >VH(SEQ ID NO.13; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.28 / SEQ ID NO.29 / SEQ IDNO.30)

[0196] QVQLVESGGGLVKPGGSLRLSCAASGFTFS SYGMS WVRQAPGKRLEWVA TISGDSIFNY

[0197] YLDTVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR QDYGSSWFAY WGQGTTVT

[0198] VSS

[0199] >VL(SEQ ID NO.14; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.31 / SEQ ID NO.32 / SEQ IDNO.33)

[0200] DIQLTQSPSSLSASVGDRVTITC SASSSVSSVH WFQQKPGTSPKRWIY GTSKLAS GVPSRF

[0201] SGSGSGTDYTLTISSLQPEDFATYYC QQWSGNPPYT FGQGTRLEIK

[0202] (II) Construction of chimeric antibodies

[0203] The coding sequences of the heavy chain variable region and light chain variable region of the murine antibody against B7-H3 were linked to the coding sequences of the heavy chain constant region of human IgG1 (SEQ ID NO.17) and the light chain constant region of human kappa (SEQ ID NO.18), respectively. The resulting coding genes were cloned into a eukaryotic expression vector, expressed, and the chimeric antibody against B7-H3 was obtained.

[0204] >Light chain (LC: SEQ ID NO.16; VL: SEQ ID NO.11; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.31 / SEQ ID NO.32 / SEQ ID NO.33; CL: SEQ ID NO.18)

[0205]

[0206] Example 7 Detection of the binding activity of anti-B7-H3 humanized antibodies to human B7-H3 overexpressing cells or human tumor cells.

[0207] CT26-B7H3 cells overexpressing human B7-H3, human esophageal squamous cell carcinoma KYSE-30 cells (Kangyuan Bochuang, Cat#: KC-2197), and human small cell lung cancer NCI-H196 cells (Kangyuan Bochuang, Cat#: KC-0454) in logarithmic growth phase were collected. After washing with PBS and resuspending, they were cultured at 2×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 50 μL / well in 96-well deep-well plates. The humanized antibody and the anti-B7-H3 control antibody Ifinatamab (Kangyuan Bochuang, Cat#: KB-1091-01) were serially diluted and incubated with the cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer. Then, the cells were incubated with the anti-human secondary antibody Goat anti-human IgG-PE at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer and resuspended. Flow cytometry was used to detect the antibody signal bound to the cells. Results are shown in the figure below. Figure 2 See 2A to 2C and Table 3.

[0208] Table 3. Results of detection of the binding activity of anti-B7-H3 humanized antibodies to human B7-H3 overexpressing cells or human tumor cells

[0209]

[0210] Example 8 Detection of the binding activity of anti-B7-H3 humanized antibody to monkey B7-H3 protein

[0211] Coat ELISA plates with 1 μg / mL cyno-B7-H3-His (Acro, Cat#: B73-C52Ha). Serially dilute the antibody to be tested, adding 50 μL / well to each well of the ELISA plate and incubating at room temperature for 1 h. Wash 5 times with PBST and blot dry. Add 50 μL / well of the diluted secondary antibody solution and incubate at room temperature for 1 h. Wash 5 times with PBST and blot dry. Add 50 μL / well of the chromogenic buffer equilibrated to room temperature and incubate in the dark for 5 min. Detect the binding signal using a microplate reader and calculate the IC50 of the antibody. Results are shown in [Figure number missing]. Figure 3 And Table 4.

[0212] Table 4. Results of detection of binding activity between anti-B7-H3 humanized antibody and monkey B7-H3 protein

[0213] Antibody EC50 (μg / mL) Ifinatamab 0.01326 H125C3Y3CV2-hIgG1 0.02579

[0214] Example 9 Detection of the internalization activity of anti-B7-H3 humanized antibody on tumor cells

[0215] KYSE-30 cells in logarithmic growth phase were collected and resuspended in FACS buffer (PBS containing 2% FBS + 2mM EDTA). 50 μL of cell suspension was added to each well of a U-bottom 96-well plate, for a total of 20,000 cells. Experimental groups were set up with no elution and elution at different times. 50 μL of diluted test antibody was added to each well to a final concentration of 10 μg / mL, and the cells were incubated on ice for 1 h. After incubation, 200 μL of FACS buffer was added to each well, and the cells were washed twice to remove the supernatant. The no-elution group was resuspended in 200 μL of FACS buffer and stored at 4°C. The 0-h elution group was eluted by adding 100 μL of elution buffer (0.05M glycine, 0.1M NaCl, pH adjusted to 3) to each well, incubating at room temperature for 6-7 min, washing once with 200 μL of FACS buffer, and then resuspending in 200 μL of FACS buffer again. The cells were stored at 4°C. Each group eluted at 0.5h, 1h, and 2h was resuspended in 200μL of FACS buffer and incubated at 37℃ for the corresponding time. The above elution steps were then repeated, and finally, the cells were resuspended in 200μL of FACS buffer and stored at 4℃. Fluorescence signals in the cells were detected by flow cytometry and the mean fluorescence intensity (MFI) was calculated. The antibody internalization rate at each time point was calculated using the following formula: Internalization rate = (MFI) / (MFI) 实验组-MFI 0h洗脱组 ) / MFI 未洗脱组 ×100%, test results are shown below. Figure 4 .

[0216] As shown in the figure, the internalization activity of the anti-B7-H3 humanized antibody is comparable to that of the control antibody.

[0217] Example 10 Preparation of hybridoma cells secreting anti-EGFR antibodies

[0218] Mice constructed in Example 2 were immunized using EGFR-mFc (Kangyuan Bochuang, Cat#: KP-1148) as an immunogen. Mice with high titers were selected, and serum was collected. After dissection, the spleen was harvested, and spleen cells were isolated. These spleen cells were fused with cultured myeloma cells to obtain hybridoma cells. The binding activity of the hybridoma cell supernatant to the antigen protein EGFR-His (Kangyuan Bochuang, Cat#: KP-1150) was detected using ELISA, yielding multiple positive hybridoma cell lines secreting anti-EGFR antibodies.

[0219] A murine antibody against EGFR was obtained from a positive hybridoma cell line and named EGFR-KD-22-0205-31A2-4 (abbreviated as "KD-22-0205" or "31A2-4"). Its variable region sequence is as follows (where the heavy chain and light chain CDRs are underlined):

[0220] EGFR-KD-22-0205-31A2-4 (Mouse Antibody)

[0221] >VH(SEQ ID NO.12; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.34 / SEQ ID NO.35 / SEQ IDNO.36)

[0222] EVHLQQSGPELVKPGASVKISCKASGYTFT DYYMN WVKQSHGKSLEWIG DINPNDGGT

[0223] SYNQKFKG KATLTVDKSSSTAYMELRRSLTSEDSAVYYCAR EILYYGNSFYFDY WGQGTT

[0224] LTVSS

[0225] >VL(SEQ ID NO.11; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.31 / SEQ ID NO.32 / SEQ IDNO.33)

[0226] RIVLTQSPGFMSASPGEKVTMTC SASSSVSSVH WFQQKSGTSPKRWIY GTSKLAS GVPA

[0227] RFSGSGSGTSYSLTISSMEAEDAATYYC QQWSGNPPYT FGGGTKLEIKRA

[0228] Example 11 Detection of the binding activity of anti-EGFR murine antibody to human EGFR-overexpressing cells

[0229] CT26-EGFR (Kangyuan Bochuang, Cat#: KC-1451) cells overexpressing human EGFR were collected during the logarithmic growth phase, washed with PBS and resuspended, then cultured at 2×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 50 μL / well in 96-well deep-well plates. The mouse antibody and anti-EGFR control antibody Cetuximab (Kangyuan Bochuang, Cat#: KA-1398) were serially diluted and incubated with the cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer (PBS + 2% FBS + 2 mM EDTA). Then, each well was incubated with either the anti-human secondary antibody Goat anti-human IgG-PE or the anti-mouse secondary antibody PE anti-mouse IgG Fc at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer and resuspended. Flow cytometry was used to detect the antibody signal binding to the cells; the results are shown in Table 5.

[0230] Table 5. Results of the detection of binding activity between anti-EGFR murine antibodies and human EGFR-overexpressing cells.

[0231] Antibody EC50 (μg / mL) Cetuximab 0.01888 31A2-4 0.056

[0232] Example 12 Detection of the blocking activity of anti-EGFR murine antibody against EGFR-EGF binding

[0233] The antibodies to be tested were serially diluted and co-incubated with 0.4 μg / ml EGFR-mFc (Kangyuan Bochuang, Cat#: KP-1148) or EGFR-hFc (Kangyuan Bochuang, Cat#: KP-1149), then incubated with ELISA plates coated with EGF-His (2 μg / ml, Acro, Cat#: EGF-H52H3), and finally co-incubated with anti-mouse secondary antibody HRP anti-mouse IgG (SIGMA, Cat#: A9309) or anti-human secondary antibody HRP anti-human IgG (SIGMA, Cat#: A0170). The binding signal was detected by an ELISA reader, and the IC50 of the antibody was calculated. The results are shown in Table 6.

[0234] Table 6. Results of detection of the blocking activity of murine anti-EGFR antibodies and control antibodies against EGFR-EGF binding.

[0235] Antibody IC50 (μg / mL) Cetuximab 0.4083 31A2-4 2.76

[0236] Example 13 Detection of affinity of murine anti-EGFR antibodies for human EGFR protein

[0237] The ForteBio Octet assay was used to detect the affinity between the anti-EGFR murine antibody and human EGFR protein. Human EGFR-His (Kangyuan Bochuang, Cat#: KP-1150) was used as the mobile phase and diluted sequentially to 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM, with simultaneous detection. The results are shown in Table 7.

[0238] Table 7. Results of the detection of affinity of murine anti-EGFR antibodies for human EGFR protein

[0239] Antibody ka(1 / Ms) kd(1 / s) KD(M) Cetuximab 1.22E+06 2.20E-03 1.80E-09 31A2-4 1.21E+06 2.52E-03 2.08E-09

[0240] Example 14 Construction of humanized and chimeric antibodies against EGFR

[0241] (I) Construction of humanized antibodies

[0242] The heavy chain and light chain variable regions of the anti-EGFR murine antibody were humanized to obtain a humanized antibody sequence. The coding sequences of the humanized antibody sequence were linked to the coding sequences of the heavy chain constant region (SEQ ID NO.17) of human IgG1 and the light chain constant region (SEQ ID NO.18) of human kappa, respectively. The resulting coding genes were cloned into a eukaryotic expression vector, expressed, and the anti-EGFR humanized antibody was obtained, named EGFR-KA-2893-H31A2-4V2-hIgG1 (abbreviated as "H31A2-4V2-hIgG1" or "KA-2893"). Its heavy chain and light chain variable region sequences and full-length heavy and light chains are as follows (where the heavy chain and light chain CDRs are underlined).

[0243] EGFR-KA-2893-H31A2-4V2-hIgG1 (humanized antibody)

[0244] >VH(SEQ ID NO.15; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.34 / SEQ ID NO.35 / SEQ IDNO.36)

[0245] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYYMN WVRQAPGQGLEWIG DINPNDG

[0246] GTSYNQKFKG RATLTVDKSTSTAYMELSSLRSEDTAVYYCAR EILYYGNSFYFDY WGQG

[0247] TTVTVSS

[0248] >VL(SEQ ID NO.14; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.31 / SEQ ID NO.32 / SEQ IDNO.33)

[0249] DIQLTQSPSSLSASVGDRVTITC SASSSVSSVH WFQQKPGTSPKRWIY GTSKLAS GVPSRF

[0250] SGSGSGTDYTLTISSLQPEDFATYYC QQWSGNPPYT FGQGTRLEIK

[0251] (II) Construction of chimeric antibodies

[0252] The coding sequences of the heavy chain variable region and light chain variable region of the anti-EGFR murine antibody were linked to the coding sequences of the heavy chain constant region of human IgG1 (SEQ ID NO.17) and the light chain constant region of human kappa (SEQ ID NO.18), respectively. The resulting coding genes were cloned into a eukaryotic expression vector, expressed, and used to obtain a chimeric antibody against EGFR.

[0253] >Light chain (LC: SEQ ID NO.16; VL: SEQ ID NO.11; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.31 / SEQ ID NO.32 / SEQ ID NO.33; CL: SEQ ID NO.18)

[0254]

[0255] Therefore, it can be seen that the chimeric antibody against B7-H3 and the chimeric antibody against EGFR have the same amino acid sequence in the light chain variable region (SEQ ID NO.11) and the same amino acid sequence in the light chain (SEQ ID NO.16); the italicized part is the human light chain CL sequence.

[0256] Example 15 Detection of the binding activity of humanized anti-EGFR antibodies to human EGFR-overexpressing cells or human tumor cells expressing EGFR.

[0257] CT26-EGFR cells overexpressing human EGFR, human esophageal squamous cell carcinoma KYSE-30 cells, and human gastric cancer MKN74 (Kangyuan Bochuang, Cat#: KC-0403) cells in logarithmic growth phase were collected. After washing with PBS and resuspending, the cells were cultured at 2×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 50 μL / well in 96-well deep-well plates. The humanized antibody and anti-EGFR control antibody Cetuximab (Kangyuan Bochuang, Cat#: KA-1398-01) were serially diluted and incubated with the cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer. Then, the cells were incubated with the anti-human secondary antibody Goat anti-human IgG-PE at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer and resuspended. Flow cytometry was used to detect the antibody signal binding to the cells; the results are shown in Table 8.

[0258] Table 8. Detection results of the binding activity of anti-EGFR humanized antibodies to human EGFR-overexpressing cells or human tumor cells.

[0259]

[0260] Example 16 Detection of the binding activity of anti-EGFR humanized antibody to monkey EGFR protein

[0261] The ELISA plate was coated with 1 μg / mL cyno-EGFR-His (Acro, Cat#: EGR-C52H1). The antibody was serially diluted and added to each well at 50 μL, incubating at room temperature for 1 h. After washing five times with PBST and patting dry, 50 μL of the diluted secondary antibody solution was added to each well, and the plate was incubated at room temperature for 1 h. After washing five times with PBST and patting dry, 50 μL of the chromogenic solution equilibrated to room temperature was added to each well, and the plate was incubated in the dark for 5 min. The binding signal was detected using a microplate reader, and the IC50 of the antibody was calculated. The results are shown in Table 9.

[0262] Table 9. Results of detection of the binding activity of anti-EGFR humanized antibody to monkey EGFR protein

[0263] Antibody EC50 (μg / mL) Cetuximab 0.1809 H31A2-4V2-hIgG1 0.2196

[0264] Example 17 Detection of the blocking activity of anti-EGFR humanized antibodies against EGFR-EGF binding.

[0265] The antibody to be tested was serially diluted and co-incubated with CT26-EGFR cells, then co-incubated with 0.05 μg / ml EGF-mFc (Acro, Cat#: EGF-H525b), and finally co-incubated with the anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307). Flow cytometry was used to detect the EGF-mFc signal bound to the cells, and the results are shown in Table 10.

[0266] Table 10. Detection of the blocking activity of anti-EGFR humanized antibodies against EGFR-EGF binding.

[0267] Antibody IC50 (μg / mL) Cetuximab 0.004961 H31A2-4V2-hIgG1 0.007692

[0268] Example 18 Assembly of B7-H3×EGFR bispecific antibody

[0269] The variable region of the light chain of the humanized antibody H125C3Y3CV2-hIgG1 against B7H3 was linked to the coding sequence of the constant region of the light chain of human kappa (SEQ ID NO.18) to obtain the common light chain of the bispecific antibody.

[0270] The heavy chain variable region (SEQ ID NO. 13) of the anti-B7-H3 humanized antibody H125C3Y3CV2-hIgG1 was linked to the coding sequence of the human heavy chain Knob CH1 and FC sequence (SEQ ID NO. 19) to obtain the coding sequence of one heavy chain (heavy chain 1) of the bispecific antibody. The heavy chain variable region (SEQ ID NO. 15) of the anti-EGFR humanized antibody H31A2-4V2-hIgG1 was linked to the coding sequence of the human heavy chain Hole CH1 and FC sequence (SEQ ID NO. 20) to obtain the coding sequence of the other heavy chain (heavy chain 2) of the bispecific antibody.

[0271] The obtained coding sequences were cloned into eukaryotic expression vectors to obtain three recombinant expression plasmids (one plasmid encoding the common light chain, one plasmid encoding heavy chain 1 targeting B7-H3, and one plasmid encoding heavy chain 2 targeting EGFR).

[0272] The HEK293F expression system was used to transiently co-transfect three plasmids to produce B7-H3×EGFR bispecific antibodies. In short, for a 1L shake flask, HEK293F cells were introduced at a rate of 1×10⁻⁶ cells / mL. 6 Inoculate at a density of 1 / mL in 250mL of culture medium and incubate at 110rpm in 5% CO2. The next day, mix the three pre-prepared expression vectors with the transfection reagent in a certain ratio and then inoculate at 2×10⁻⁶. 6 At a cell density of 10 cells / mL, the transfection complex was added to the cells. After 24 hours, nutrients and DNA inhibitors were added. 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 detected by SDS-PAGE electrophoresis, and the antibody concentration was detected by Nanodrop.

[0273] The bispecific antibody B7-H3×EGFR, B7H3xEGFR-KA-3123-H125C3Y3CV2xH31A2-4V2-hIgG1 (abbreviated as "H125C3Y3CV2xH31A2-4V2-hIgG1" or "KA-3123"), was obtained.

[0274] The heavy chain (HC) and light chain (LC) sequences of the bispecific antibody KA-3123 are as follows; italics indicate constant regions. KA-3123-H125C3Y3CV2xH31A2-4V2-hIgG1 (B7-H3×EGFR bispecific antibody)

[0275] >Heavy chain 1 (HC1: SEQ ID NO.23; VH: SEQ ID NO.13; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.28 / SEQ ID NO.29 / SEQ ID NO.30; CH[Knob]: SEQ ID NO.19)

[0276]

[0277] >Heavy chain 2 (HC2: SEQ ID NO.24; VH: SEQ ID NO.15; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.34 / SEQ ID NO.35 / SEQ ID NO.36; CH[Hole]: SEQ ID NO.20)

[0278]

[0279] >Common light chain (LC: SEQ ID NO.25; VL: SEQ ID NO.14; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.31 / SEQ ID NO.32 / SEQ ID NO.33; CL: SEQ ID NO.18)

[0280]

[0281] The domains contained in the bispecific antibody KA-3123 and their sequence numbers are shown in Table 11.

[0282] Table 11. Bispecific antibody KA-3123 containing a common light chain and B7-H3×EGFR

[0283]

[0284] Example 19 Detection of affinity between anti-B7-H3 or anti-EGFR humanized antibodies, B7-H3×EGFR bispecific antibodies and human B7-H3 and EGFR proteins

[0285] The ForteBio Octet was used to detect the affinity of the antibody for human B7-H3 and EGFR proteins. The mobile phase consisted of human B7H3-His (Kangyuan Bochuang, Cat#: KP-1114) and human EGFR-His (Kangyuan Bochuang, Cat#: KP-1150), diluted sequentially to 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM. The results are shown in Table 12.

[0286] Table 12. Detection results of affinity between different antibodies and human B7-H3 and EGFR proteins

[0287]

[0288]

[0289] Example 20 Detection of the binding activity of anti-B7-H3 or anti-EGFR humanized antibodies, B7-H3×EGFR bispecific antibodies with human EGFR, human B7-H3 overexpressing cells and human tumor cells.

[0290] Logarithmic growth phase CT26-EGFR cells, CT26-B7H3 cells, human cervical cancer cells MS751 (Kangyuan Bochuang, Cat#: KC-0948, expressing EGFR and B7-H3), human lung cancer cells NCI-H661 (Kangyuan Bochuang, Cat#: KC-0515, expressing only B7-H3, not EGFR), and human non-small cell lung cancer cells NCI-H1568 (Kangyuan Bochuang, Cat#: KC-0369, expressing EGFR and B7-H3) were collected, washed with PBS and resuspended, and cultured at 2×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 50 μL / well in 96-well deep-well plates. The bispecific antibody KA-3123, the parent antibody KA-2893-H31A2-4V2-hIgG1 (anti-EGFR), KA-2802-H125C3Y3CV2-hIgG1 (anti-B7H3), and control antibodies Cetuximab (anti-EGFR) and lfinatamab (anti-B7H3) were serially diluted and incubated with the cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL of FACS buffer (PBS + 2% FBS + 2 mM EDTA). Then, the cells were incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09) at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL of FACS buffer and resuspended. Flow cytometry was used to detect antibody signals bound to cells; the results are shown in Tables 13 to 15. Figure 5 5A to 5E in the series.

[0291] Table 13. Detection results of the binding activity of different antibodies to CT26-B7H3 cells

[0292] Antibody EC50 (μg / mL) Ifinatamab 0.132 H125C3Y3CV2-hIgG1 0.06116 H125C3Y3CV2xH31A2-4V2-hIgG1 0.05797 IgG1 Isotype (Kangyuan Bochuang, Cat#: KP-2001) N / A

[0293] Table 14. Results of detection of the binding activity of different antibodies to CT26-EGFR cells

[0294] Antibody EC50 (μg / mL) Cetuximab 0.009767 H31A2-4V2-hIgG1 0.008433 H125C3Y3CV2xH31A2-4V2-hIgG1 11.82 IgG1 Isotype N / A

[0295] Table 15. Detection results of the binding activity of different antibodies to antigen-positive tumor cells.

[0296]

[0297]

[0298] Example 21 Detection of the binding activity of anti-B7-H3 or anti-EGFR humanized antibodies, B7-H3×EGFR bispecific antibodies to human or monkey EGFR or B7-H3 proteins.

[0299] ELISA plates were coated with 1 μg / mL B7H3-His (Kangyuan Bochuang, Cat#: KP-1114), cyno-B7H3-His (Acro, Cat#: B73-C52Ha), EGFR-His, and cyno-EGFR-His. The antibodies were serially diluted and added to each well at 50 μL, incubating at room temperature for 1 h. After washing five times with PBST and patting dry, 50 μL of the diluted secondary antibody solution was added to each well, incubating at room temperature for 1 h. After washing five times with PBST and patting dry, 50 μL of the chromogenic solution equilibrated to room temperature was added to each well, and the plates were incubated in the dark for 5 min. The binding signal was detected using a microplate reader, and the IC50 of the antibodies was calculated. The results are shown in Table 16. Figure 6 6A to 6D in the middle.

[0300] Table 16. Detection results of the binding activity of different antibodies to human or monkey EGFR or B7-H3 proteins

[0301]

[0302] Example 22 Detection of the blocking activity of anti-EGFR humanized antibody and B7-H3×EGFR bispecific antibody against EGFR-EGF binding.

[0303] The antibody to be tested was serially diluted and co-incubated with NCI-H1568 cells, then co-incubated with 0.05 μg / ml EGF-mFc, and finally co-incubated with the anti-mouse secondary antibody FITC anti-mouse IgG2a (Biolegend, Cat#: 407106). Flow cytometry was used to detect the EGF-mFc signal bound to the cells, and the MFI was calculated and normalized to the group without primary antibody incubation. The results are shown in Table 17. Figure 7 .

[0304] Table 17. Detection results of the blocking activity of anti-EGFR humanized antibody and B7-H3×EGFR bispecific antibody against EGFR-EGF binding.

[0305] Antibody IC50 (μg / mL) Maximum blocking rate (%) Cetuximab 0.006681 91.65 H31A2-4V2-hIgG1 0.01507 90.91 H125C3Y3CV2xH31A2-4V2-hIgG1 N / A 24.79

[0306] Example 23 Detection of the binding activity of B7-H3×EGFR bispecific antibody against EGFR, B7-H3 family proteins or EGFR, B7-H3 proteins from different species.

[0307] To detect whether the B7-H3×EGFR bispecific antibody has binding activity with proteins of the same family or other species as EGFR or B7-H3, the following ELISA or flow cytometry assays were performed.

[0308] (1) Detection of the binding activity of B7-H3×EGFR bispecific antibody against EGFR homofamily antigens

[0309] CT26, CHOK1, and Ba / F3 cells (CT26-ERBB2, Kangyuan Bochuang, Cat#: KC-1389; CHOK1-ERBB3, Kangyuan Bochuang, Cat#: KC-1511; Ba / F3-ERBB4, Kangyuan Bochuang, Cat#: KC-2410) overexpressing EGFR family proteins ERBB2, ERBB3, and ERBB4 were collected during logarithmic growth phase. Cells were washed with PBS, resuspended, and cultured at 2 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 50 μL per well in 96-well deep-well plates. Serial dilutions of the target antibiotic and isotype control were performed and incubated with cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer. Cells were then incubated with the anti-human secondary antibody (Goat anti-human IgG-PE) at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer and resuspended. Flow cytometry was used to detect antibody signals bound to the cells. Results are shown below. Figure 8 8A to 8C in the middle.

[0310] (2) Detection of the binding activity of B7-H3×EGFR bispecific antibody against different species antigens of EGFR

[0311] ELISA plates were coated with 1 μg / mL rat and mouse EGFR protein (Recombinant Rat EGFR Protein, SinoBiological, Cat#: 80100-R08H; Mouse EGFR Protein, Acro, Cat#: EGR-M5224). The antibodies were serially diluted and added to each well at 50 μL, and incubated at room temperature for 1 h. After washing five times with PBST and patting dry, 50 μL of diluted secondary antibody solution was added to each well, and the plates were incubated at room temperature for 1 h. After washing five times with PBST and patting dry, 50 μL of chromogenic buffer (equilibrated to room temperature) was added to each well, and the plates were incubated in the dark for 5 min. The binding signal was detected using a microplate reader. Results are shown below. Figure 9 9A to 9B in the middle.

[0312] (3) Detection of the binding activity of B7-H3×EGFR bispecific antibody against different species antigens of B7-H3

[0313] 293T cells overexpressing mouse B7-H3 and their corresponding 293T blast cells (293T-mouse-B7H3, Kangyuan Bochuang, Cat#: KC-2425; 293T, Kangyuan Bochuang, Cat#: KC-0159) were collected in logarithmic growth phase, washed with PBS and resuspended, then incubated at 2×10⁻⁶ ppm. 4 Cells were seeded at a density of 50 μL per cell per well in 96-well deep-well plates. Serial dilutions of the target antibiotic and isotype control were performed and incubated with cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer. Cells were then incubated with the anti-human secondary antibody Goat anti-human IgG-PE at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer and resuspended. Flow cytometry was used to detect antibody signals bound to the cells.

[0314] The ELISA plate was coated with 1 μg / mL Rat B7-H3 protein (Recombinant Rat B7-H3 Protein, SinoBiological, 80380-R08H). The antibody was serially diluted and added to each well at 50 μL, incubating at room temperature for 1 h. The plates were washed five times with PBST and blotted dry. 50 μL of the diluted secondary antibody solution was added to each well, and the plates were incubated at room temperature for 1 h. The plates were then washed five times with PBST and blotted dry. 50 μL of the chromogenic buffer (equilibrated to room temperature) was added to each well, and the plates were incubated in the dark for 5 min. The binding signal was detected using a microplate reader.

[0315] Test results are shown Figure 10 10A to 10C in the range.

[0316] (4) Detection of the binding activity of B7-H3×EGFR bispecific antibody against B7-H3 family antigens

[0317] Use 1 μg / mL of B7-H3 family proteins B7-H3-mFc, PD-L1-mFc (Kangyuan Bochuang, Cat#: KP-1008), ICOS-L-His (Kangyuan Bochuang, Cat#: KP-1108), B7-H4-mFc (Kangyuan Bochuang, Cat#: KP-1279), Human B7-H5-His (Acro, Cat#: B75-H52H0), Human B7-H6-His (Acro, Cat#: B76-H52H8), Human PD-L2-His (Acro, Cat#: PD2-H5220), Human B7-1-Fc (Acro, Cat#: B71-H52A4), Human B7-2-His (Acro, Cat#: CD6-H5223) or 2% BSA was used to coat ELISA plates. The antibody was serially diluted and added to each well at 50 μL, and incubated at room temperature for 1 hour. The plates were washed five times with PBST and patted dry. 50 μL of the diluted secondary antibody solution was added to each well, and the plates were incubated at room temperature for 1 hour. The plates were then washed five times with PBST and patted dry. 50 μL of the chromogenic buffer (equilibrated to room temperature) was added to each well, and the plates were incubated in the dark for 5 minutes. The binding signal was detected using a microplate reader. Results are shown below. Figure 11 11A to 11B in the middle.

[0318] The results are summarized in Table 18.

[0319] Table 18. Detection results of the binding activity of B7-H3×EGFR bispecific antibody against EGFR, B7-H3 family proteins or different species proteins of EGFR and B7-H3.

[0320]

[0321] Example 24 Detection of internalization activity of anti-B7-H3 humanized antibody and B7-H3×EGFR bispecific antibody on EGFR and / or B7-H3-expressing tumor cells.

[0322] MS751, NCI-H661, and NCI-H1568 cells in logarithmic growth phase were collected and resuspended in FACS buffer (PBS containing 2% FBS + 2mM EDTA). 50 μL of cell suspension was added to each well of a U-bottom 96-well plate, for a total of 20,000 cells. Experimental groups included no elution and elution at different times. 50 μL of diluted test antibody was added to each well to a final concentration of 1 μg / mL, and the cells were incubated on ice for 1 h. After incubation, 200 μL of FACS buffer was added to each well, and the cells were washed twice to remove the supernatant. The no-elution group was resuspended in 200 μL of FACS buffer and stored at 4°C. Elution was performed on the 0h elution group by adding 100 μL of elution buffer (0.05 M glycine, 0.1 M NaCl, pH adjusted to 3) to each well, incubating at room temperature for 6-7 min, washing once with 200 μL of FACS buffer, resuspending with another 200 μL of FACS buffer, and storing at 4°C. Elution groups at 0.5h, 1h, and 2h were resuspended with 200 μL of FACS buffer and incubated at 37°C for the corresponding durations, followed by the same elution steps, and finally resuspended with 200 μL of FACS buffer and stored at 4°C. Fluorescence signals in the cells were detected by flow cytometry and the mean fluorescence intensity (MFI) was calculated. The antibody internalization rate at each time point was calculated using the following formula: Internalization rate = (MFI) / (MFI) 实验组 -MFI0 h洗脱组 ) / MFI 未洗脱组 ×100%, test results are shown below. Figure 12 12A to 12C in the range.

[0323] The results showed that in cells expressing EGFR, the internalization rate of the bispecific antibody was higher than that of the corresponding humanized anti-B7-H3 antibody. In cells without EGFR expression, the internalization rate of the bispecific antibody was comparable to that of the corresponding humanized anti-B7-H3 antibody. This indicates that although the EGFR antibody arm in the bispecific antibody has low antigen-binding activity, it effectively promotes the internalization of the bispecific antibody.

[0324] Example 25 Preparation of antibody-drug conjugates (ADCs)

[0325] According to Example 5 of patent publication WO2025011419A1, the linker-drug molecule LD38 was prepared, and according to Example 18, LD38 was conjugated to Human IgG1 isotype, kappa (Kangyuan Bochuang, Cat#: KP-2001) with a drug-antibody ratio (DAR) of 8 to obtain an ADC (hereinafter referred to as hIgG1-LD38), LD38 was conjugated to Ifinatamab with DAR4 to obtain an ADC (hereinafter referred to as Ifinatamab-LD38), and LD38 was conjugated to H125C3Y3CV2xH31A2-4V2-hIgG1 with DAR8 to obtain an ADC (hereinafter referred to as KA-3123-LD38). The antibody-drug conjugate DS-7300 was prepared according to patent CN109081871B.

[0326] Example 26 Detection of ADC binding activity with EGFR and / or B7-H3 expressing cells

[0327] Human non-small cell lung cancer (NSCLC) cells NCI-H322, NCI-H661, and NCI-H1568 in the logarithmic growth phase were collected, washed with PBS, resuspended, and cultured at 2×10⁻⁶. 4 Cells were seeded at a density of 50 μL / well in 96-well deep-well plates. The test antibody and ADC were serially diluted and incubated with the cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer (PBS + 2% FBS + 2 mM EDTA). Then, the cells were incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09) at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer and resuspended. Flow cytometry was used to detect the antibody signal bound to the cells. Results are shown in [Figure number missing]. Figure 13 13A to 13C in the middle.

[0328] The results showed that, compared with the B7-H3×EGFR bispecific antibody itself, the ADC obtained by conjugating the bispecific antibody with a small molecule drug had a slightly decreased binding ability to cells expressing the antigen, but it was still stronger than the control ADC.

[0329] Example 27 Detection of ADC internalization activity in EGFR and / or B7-H3 expressing cells

[0330] MS751, NCI-H1568, and MFE-280 cells (Kangyuan Bochuang, Cat#: KC-0664) in logarithmic growth phase were collected and resuspended in FACS buffer (PBS containing 2% FBS + 2mM EDTA). 50 μL of cell suspension was added to each well of a U-bottom 96-well plate, for a total of 20,000 cells. Experimental groups included no elution and elution at different times. 50 μL of diluted test antibody was added to each well to a final concentration of 1 μg / mL, and the cells were incubated on ice for 1 h. After incubation, 200 μL of FACS buffer was added to each well, and the cells were washed twice to remove the supernatant. The no-elution group was resuspended in 200 μL of FACS buffer and stored at 4°C. Elution was performed on the 0h elution group by adding 100 μL of elution buffer (0.05 M glycine, 0.1 M NaCl, pH adjusted to 3) to each well, incubating at room temperature for 6-7 min, washing once with 200 μL of FACS buffer, resuspending with another 200 μL of FACS buffer, and storing at 4°C. Elution groups at 0.5h, 1h, and 2h were resuspended with 200 μL of FACS buffer and incubated at 37°C for the corresponding durations, followed by the same elution steps, and finally resuspended with 200 μL of FACS buffer and stored at 4°C. Fluorescence signals in the cells were detected by flow cytometry and the mean fluorescence intensity (MFI) was calculated. The antibody internalization rate at each time point was calculated using the formula: Internalization rate = (MFI) / (MFI) 实验组 -MFI0 h洗脱组 ) / MFI 未洗脱组 ×100%, results are shown below Figure 14 14A to 14C in the middle.

[0331] The results showed that the internalization rate of the ADC was not significantly different from that of the corresponding naked antibody, but was stronger than that of the control antibody and the control ADC.

[0332] Example 28 Detection of the cytotoxic activity of ADCs against human tumor cells expressing EGFR and / or B7-H3

[0333] Tumor cells in logarithmic growth phase were collected and seeded at 2000 cells / 180 μL / well in 96-well plates. The test antibody and ADC were serially diluted, with 20 μL added to each well. A negative control group without ADC was also included. Cells were cultured for 5 days. After 5 days, an equal volume of pre-equilibrated room temperature solution was added to each well. 2.0 (Vazyme, Cat#: DD1101-01) reagent was used, and the cells were lysed by shaking for 30 min. The microplate readings were taken using a microplate reader, and the cell viability of the experimental group was calculated using the negative control group as a baseline. Results are shown below. Figure 15 15A to 15F in the series.

[0334] Example 29 Detection of the bystander effect using B7-H3×EGFR ADC

[0335] Raji cells overexpressing EGFR and B7-H3 (Raji-EGFR-B7H3, Kangyuan Bochuang, Cat#: KC-4394) and Raji cells overexpressing luciferase (Raji-Luc2, Kangyuan Bochuang, Cat#: KC-2457) in logarithmic growth phase were collected and seeded in 96-well cell culture plates. Seeding conditions were as follows: 2000 cells / well of each cell type individually, or 1000 cells / well of each type mixed in one well, with 180 μL of culture medium per well. Serially diluted ADCs were added to each well (20 μL), and a negative control group without ADC was included. Cells were cultured for 5 days. After 5 days, an equal volume of pre-equilibrated room temperature culture medium was added to the wells containing only Raji-EGFR-B7H3 cells. 2.0 Reagent, and lyse by shaking. Add an equal volume of pre-equilibrated to room temperature reagent to the wells containing Raji-Luc2 cells (including wells with Raji-Luc2 cultured alone and wells with Raji-EGFR-B7H3 culture). Reagent (Vazyme, Cat#: DD1204-01) was used, and cells were lysed by shaking. Cell viability was measured using a microplate reader, and the viability of cells in the experimental group was calculated using the negative control group as a baseline. Results are shown below. Figure 16 16A to 16C in the middle.

[0336] In Raji cells overexpressing EGFR and B7-H3, the bispecific ADC KA-3123-LD38, which targets both EGFR and B7-H3, and the control ADCs DS-7300 and Ifinatamab-LD38, which target only B7-H3, all exhibited good cytotoxic activity. In Raji-Luc2 cells lacking EGFR and B7-H3 expression, none of the ADCs showed good cytotoxic activity; the single or bispecific ADCs targeting B7-H3 showed consistent activity with the isotype control ADC hIgG1-LD38, which had no targeting effect. Under conditions of co-culture of Raji-EGFR-B7H3 and Raji-Luc2 cells, the bispecific ADCs targeting both EGFR and B7-H3 and the single specific ADCs targeting only B7-H3 showed significantly enhanced cytotoxic activity against Raji-Luc2 cells, indicating a bystander killing effect.

[0337] Example 30 Antitumor activity of B7-H3×EGFR bispecific antibody or ADC in a BALB / cnude mouse model subcutaneously inoculated with human gastric cancer cells MKN-45

[0338] Collect MKN-45 (Kangyuan Bochuang, Cat#: KC-0412) cells in logarithmic growth phase at a concentration of 2 × 10⁻⁶. 6 The tumor was injected subcutaneously into the right shoulder area of ​​6-8 week old female BALB / c nude mice (Yaokang Biotechnology, Cat#: D000521) at a density of 0.2 mL / mice. The inoculation was performed when the average tumor volume reached approximately 190 mm². 3 Mice were then randomly divided into four groups of six mice each, based on tumor volume and body weight. Drug administration began immediately after grouping, with a single dose administered. The grouping start date was considered day 0. After administration, mouse body weight and tumor volume were measured twice weekly. The formula TV = 1 / 2 × a × b was used. 2 Calculate the tumor volume. Where 'a' is the long axis of the tumor and 'b' is the short axis. Mouse body weight and tumor volume results are shown below. Figure 17 .

[0339] In MKN-45 cells, both EGFR and B7-H3 were expressed at moderate levels. As shown in the figure, compared with the control group (G1, IgG1 isotype), DS-7300 (G2), KA-3123 (G3), and KA-3123-LD38 (G4) all exhibited certain antitumor activity in vivo. On day 25 after administration, KA-3123-LD38 (G4) showed a more significant tumor-suppressive effect than other test drugs. During the experiment, all groups experienced similar levels of weight loss.

[0340] Example 31 Antitumor activity of B7-H3×EGFR bispecific antibody or ADC in a BALB / cnude mouse model subcutaneously inoculated with human pharyngeal squamous cell carcinoma FaDu.

[0341] FaDu (Kangyuan Bochuang, Cat#: KC-0761) cells in logarithmic growth phase were collected at a concentration of 3 × 10⁻⁶. 6 0.2 mL / mouse cells were injected subcutaneously into the right shoulder area of ​​6-8 week old female BALB / c nude mice (Yaokang Biotechnology, Cat#: D000521). The inoculation was performed when the average tumor volume reached approximately 164 mm². 3 Mice were then randomly divided into four groups of six mice each, based on tumor volume and body weight. Drug administration began immediately after grouping, with a single dose administered. The grouping start date was considered day 0. After administration, mouse body weight and tumor volume were measured twice weekly. The formula TV = 1 / 2 × a × b was used. 2 Calculate the tumor volume. Where 'a' is the long axis of the tumor and 'b' is the short axis. Mouse body weight and tumor volume results are shown below. Figure 18 .

[0342] In FaDu cells, EGFR was highly expressed, while B7-H3 were moderately expressed. As shown in the figure, compared with the control group (G1, IgG1 isotype), DS-7300 (G2), KA-3123 (G3), and KA-3123-LD38 (G4) all exhibited certain antitumor activity in vivo. On day 20 after administration, at a concentration of 2 mg / kg, the tumor inhibition rate of the G4 test drug was 89.1%, indicating that this group of test drugs had strong efficacy. No significant decrease in body weight was observed in any group of mice during the experiment.

[0343] Example 32 Antitumor activity of B7-H3×EGFR bispecific antibody or ADC in a BALB / cnude mouse model subcutaneously inoculated with human lung cancer cells EBC-1

[0344] Collect EBC-1 (Kangyuan Bochuang, Cat#: KC-0761) cells in logarithmic growth phase at a concentration of 2 × 10⁻⁶. 6 0.2 mL / mouse cells were injected subcutaneously into the right shoulder area of ​​6-8 week old female BALB / c nude mice (Yaokang Biotechnology, Cat#: D000521). The inoculation was performed when the average tumor volume reached approximately 193 mm². 3 Mice were then randomly divided into four groups of six mice each, based on tumor volume and body weight. Drug administration began immediately after grouping, with a single dose administered. The grouping start date was considered day 0. After administration, mouse body weight and tumor volume were measured twice weekly. The formula TV = 1 / 2 × a × b was used. 2 Calculate the tumor volume. Where 'a' is the long axis of the tumor and 'b' is the short axis. Mouse body weight and tumor volume are shown in [reference needed]. Figure 19 .

[0345] In EBC-1 cells, EGFR was highly expressed, while B7-H3 were moderately expressed. As shown in the figure, compared with the control group (G1, IgG1 isotype), DS-7300 (G2) and KA-3123 (G3) showed no significant antitumor activity, while only KA-3123-LD38 (G4) showed significant antitumor activity in vivo. During the experiment, the body weight of mice in each group did not decrease significantly.

[0346] Example 33 Fc mutation and sequence modification of B7-H3×EGFR bispecific antibody

[0347] The B7H3×EGFR bispecific antibody KA-3123 exhibited multiple elution peaks during HIC-HPLC. To ensure antibody homogeneity, the EGFR-targeting heavy chain and common light chain in KA-3123 were mutated. Simultaneously, the Fc region was mutated with L234A / L235A mutations to remove the ADCC function of the bispecific antibody. Following these requirements, one mutated EGFR heavy chain (hole chain) and one mutated common light chain were reconstructed. Referring to Example 5, another B7-H3×EGFR bispecific antibody with the effector function eliminated was assembled, named B7H3xEGFR-KA-A1816-H125C3Y3CV2xH31A2Y4CV8-CLC129-hIgG1m39x40 (abbreviated as "H125C3Y3CV2xH31A2Y4CV8-CLC129-hIgG1m39x40" or "KA-A1816"), and expressed and purified.

[0348] The heavy chain (HC) and light chain (LC) sequences of the bispecific antibody KA-A1816 are as follows; italics indicate constant regions. B7H3xEGFR-KA-A1816-H125C3Y3CV2xH31A2Y4CV8-CLC129-hIgG1m39x40

[0349] >Heavy chain 1 (HC1: SEQ ID NO.26; VH: SEQ ID NO.13; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.28 / SEQ ID NO.29 / SEQ ID NO.30; CH[Knob; LALA]: SEQ ID NO.21)

[0350]

[0351] >Heavy chain 2 (HC2: SEQ ID NO.27; VH: SEQ ID NO.37; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.34 / SEQ ID NO.38 / SEQ ID NO.36; CH[Hole; LALA]: SEQ ID NO.22)

[0352]

[0353]

[0354] >Common light chain (LC: SEQ ID NO.39; VL: SEQ ID NO.40; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.31 / SEQ ID NO.32 / SEQ ID NO.41; CL: SEQ ID NO.18)

[0355]

[0356] The domains and their sequence numbers contained in the B7-H3×EGFR bispecific antibody KA-A1816 are shown in Table 19.

[0357] Table 19. B7-H3×EGFR bispecific antibody KA-A1816 containing a common light chain

[0358]

[0359] Example 34 Physicochemical properties analysis of B7-H3×EGFR bispecific antibody

[0360] The following tests were performed on the B7-H3×EGFR bispecific antibodies KA-A1816 or KA-3123.

[0361] (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0362] Take 6 μg of the antibody sample to be tested, of which 4 μg is used for the detection of the reduced sample and 2 μg is used for the detection of the non-reduced sample, both prepared into 10 μL systems. Add 2.5 μL of 6×SDS Loading Buffer (TransGold, Cat#: DL101-02) to the reduced sample, add 2.5 μL of non-Reducing Loading Buffer (Kangwei Century, Cat#: CW0028S) to the non-reduced sample, and add 5 μL of iodoacetamide (Wokai Biotechnology, Cat#: D16141). Denature the reduced sample and the non-reduced sample at 100℃ for 15 min and 5 min, respectively. Take 10 μL of each sample and add it to a pre-prepared polyacrylamide gel (GenScript, Cat#: M00652) for electrophoresis. Maintain a constant voltage of 80V, run the gel to 1 / 3 of its length, then adjust the constant voltage to 120V and continue for about 40 min. Remove the gel and immerse it in Coomassie Brilliant Blue developing solution (Beyotime, Cat#: P0004M) for 1.5 hours. After staining, wash off the Coomassie Brilliant Blue developing solution with water, then destain overnight in water. Photograph the gel image. Results are shown below. Figure 20 20A in the middle.

[0363] (2) Size exclusion chromatography (SEC-HPLC)

[0364] The bispecific antibody at a concentration greater than 1 mg / mL was analyzed using a 1260 HPLC system. The sample loading volume was set to 10 μL, the flow rate to 0.6 mL / min, and the analysis time to 30 min. The absorbance at 280 nm was measured at 23 ± 0.8 °C to determine the amount of these substances, and the SEC chromatogram was recorded. The bispecific antibody showed a distinct monomer peak, and the peak area was greater than 95%, indicating that the bispecific antibody did not exhibit significant aggregation. The detection results are shown below. Figure 20 20B in the middle.

[0365] (3) Hydrophobic interaction high performance liquid chromatography (HIC-HPLC)

[0366] A 20 μL volume of analyte was injected onto a 4.6 mm × 10 cm hydrophobic interaction column with a spherical silica matrix of 5 μm particle size. Aqueous phosphate-sulfate buffer (APB) was used as mobile phase A, and aqueous phosphate buffer (APB) as mobile phase B, at a flow rate of 0.8 mL / min. The absorbance of the eluent was continuously monitored at 280 nm. The mobile phase gradient was as follows: 0–3 min, 0% B; 3–4.5 min, 0%–20% B; 4.5–9.5 min, 20%–60% B; 4.5–14.5 min, 60%–100% B; 14.5–16 min, 100% B; 16–18.5 min, 100%–0% B; 18.5–25 min, 0% B. Different hydrophobic components were separated on the column and eluted at different retention times. The amounts of these substances were measured by monitoring the peak absorbance at 280 nm. Test results are shown Figure 20 20C in.

[0367] (4) Tm determination

[0368] The Tm value of the antibody was determined using a 7500 Fast Real-time PCR System, as detailed below. Protein Thermal Shift Dye Kit was used for 125-fold dilution. TM For the reagent, mix the diluted test reagent and 0.5 mg / mL of the test antibody at a 1:7 ratio, and add MicroAmp. TM The reaction was performed in a 96-well optical plate and sealed with a sealing film. The plate was tested according to the recommended procedure, and trend graphs were plotted using Protein Thermal Shift Software 1.4 to obtain the Tm values. The results are shown in Table 20.

[0369] Table 20. Tm values ​​of B7-H3×EGFR bispecific antibodies

[0370] Antibody Tm (°C) H125C3Y3CV2xH31A2-4V2-hIgG1 62.55 H125C3Y3CV2xH31A2Y4CV8-CLC129-hIgG1m39x40 62.3

[0371] Example 35 Thermostability assay of modified B7-H3×EGFR bispecific antibody

[0372] The B7-H3×EGFR bispecific antibody KA-A1816 was treated under different thermostability test conditions for corresponding times or cycles. The treated antibody and the untreated antibody were then subjected to size exclusion chromatography (SEC-HPLC) and protein concentration detection. Test conditions included: 25℃ for 24 h; 5 freeze-thaw cycles; 4 h in an acidic environment (pH=3.6); 4 h in an alkaline environment (pH=9.0); 1 week at 40℃; and 2 weeks at 40℃. Concentration was detected by Nanodrop, and purity was detected by SEC-HPLC. The results are shown in Table 21. The results indicate that the bispecific antibody remained stable under various test conditions.

[0373] Table 21. Results of thermostability test for bispecific antibodies

[0374]

[0375] Example 36 Detection of affinity of modified B7-H3×EGFR bispecific antibody for human EGFR and B7-H3 proteins

[0376] ForteBio Octet assays were used to detect the affinity of KA-A1816 for EGFR and B7-H3 proteins. Human EGFR-His (Kangyuan Bochuang, Cat#: KP-1150) and human B7H3-His (Kangyuan Bochuang, Cat#: KP-1114) were used as mobile phases, diluted sequentially to 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM, respectively, and were detected simultaneously. According to the results, the affinity of the B7-H3×EGFR bispecific antibody KA-A1816 did not change significantly after sequence modification. The results are shown in Table 22.

[0377] Table 22. Detection results of affinity of the modified B7-H3×EGFR bispecific antibody for human EGFR and B7-H3 proteins.

[0378]

[0379] Example 37 Detection of the binding activity of B7-H3×EGFR bispecific antibody against human EGFR and B7-H3 proteins before and after modification

[0380] Coat ELISA plates with 1 μg / mL EGFR-His or B7-H3-His. Serially dilute the antibody to be tested, adding 50 μL / well to each well of the ELISA plate and incubating at room temperature for 1 h. Wash 5 times with PBST and blot dry. Add 50 μL / well of secondary antibody solution and incubate at room temperature for 1 h. Wash 5 times with PBST and blot dry. Add 50 μL / well of chromogenic buffer equilibrated to room temperature and incubate in the dark for 5 min. Detect the binding signal using a microplate reader and calculate the EC50 of the antibody. Results are shown in the figure. Figure 21 Tables 21A to 21B and 23 are included.

[0381] Table 23. Detection results of the binding activity of B7-H3×EGFR bispecific antibody against human EGFR and B7-H3 proteins before and after modification.

[0382]

[0383] Example 38 Detection of the binding activity of B7-H3×EGFR bispecific antibody to EGFR and / or B7-H3 overexpressing cells before and after modification

[0384] CT26-EGFR (Kangyuan Bochuang, Cat#: KC-1451) cells overexpressing EGFR, human lung cancer cells NCI-H661 (Kangyuan Bochuang, Cat#: KC-0515), human non-small cell lung cancer cells NCI-H1568 (Kangyuan Bochuang, Cat#: KC-0369), CHOK1-cyno-EGFR (Kangyuan Bochuang, Cat#: KC-1165) overexpressing cynomolgus monkey EGFR, and CHOK1-cyno-B7H3 (Kangyuan Bochuang, Cat#: KC-5660) overexpressing cynomolgus monkey B7-H3 were collected in logarithmic growth phase, washed with PBS and resuspended, and cultured at 2×10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 50 μL / well in 96-well deep-well plates. The test antibody was serially diluted and incubated with the cells at 4°C for 1 h. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer (PBS + 2% FBS + 2 mM EDTA). Then, each well was incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09) at 4°C for 50 min. After centrifugation, the supernatant was removed, and each well was washed three times with 300 μL FACS buffer and resuspended. Flow cytometry was used to detect the antibody signal bound to the cells. Results are shown in [Figure number missing]. Figure 22 22A to 22E and Table 24.

[0385] Table 24. Results of the detection of binding activity between B7-H3×EGFR bispecific antibody and EGFR and / or B7-H3 overexpressing cells before and after modification.

[0386]

[0387] Example 39 Detection of internalization activity of B7-H3×EGFR bispecific antibody in human tumor cells before and after modification

[0388] VHH Deep Red630 (AlpVHHs, Cat#: 023-101-014) is a nanobody that is coupled with a pH-sensitive fluorescent probe and can specifically recognize human IgG. It is essentially non-fluorescent in a neutral extracellular environment, but emits a bright fluorescent signal in the acidic environment of endosomes and lysosomes.

[0389] Human non-small cell lung cancer (NSCLC) cells (NCI-H1568) in logarithmic growth phase were collected and seeded at a density of 20,000 cells / 50 μL / well in 96-well cell culture plates and cultured overnight. A 4× antibody solution was prepared to achieve a final antibody concentration of 50 nM; the solution was then protected from light. Prepare VHH secondary antibody working solution to achieve a final concentration of 150 nM. Mix the antibody and secondary antibody in equal volumes and incubate at room temperature in the dark for 30 min to form a labeling complex. Add 50 μL of the labeling complex to a 96-well plate and continue culturing for 24 h. Collect cells from the 96-well plate and detect fluorescence signals in the cells using flow cytometry. The results are shown in the figure. Figure 23 .

[0390] The results showed that the internalization activity of the modified bispecific antibody molecules did not change significantly.

[0391] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. A polypeptide, said polypeptide comprising the following amino acid sequence: (i) the amino acid sequence shown in SEQ ID NO.31; the amino acid sequence shown in SEQ ID NO.32; and the amino acid sequence shown in SEQ ID NO.33; or (ii) the amino acid sequence shown in SEQ ID NO.31; the amino acid sequence shown in SEQ ID NO.32; and the amino acid sequence shown in SEQ ID NO.

41.

2. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 1.

3. The use of the polypeptide of claim 1 or the nucleic acid molecule of claim 2 in the construction of antibodies; Preferably, the antibody is a bispecific antibody.

4. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising the polypeptide of claim 1; Preferably, the antibody or its antigen-binding fragment comprises the polypeptide of claim 1 as a light chain variable region or light chain; Preferably, the antibody or its antigen-binding fragment is an IgG-like antibody or an antibody in the form of scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody or its antigen-binding fragment respectively comprises heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3) as shown below: (1) HCDR1 containing the amino acid sequence shown in SEQ ID NO.28, HCDR2 containing the amino acid sequence shown in SEQ ID NO.29, HCDR3 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.33; (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO.34, HCDR2 containing the amino acid sequence shown in SEQ ID NO.35, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.33; (3) HCDR1 containing the amino acid sequence shown in SEQ ID NO.28, HCDR2 containing the amino acid sequence shown in SEQ ID NO.29, HCDR3 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.41; (4) HCDR1 containing the amino acid sequence shown in SEQ ID NO.34, HCDR2 containing the amino acid sequence shown in SEQ ID NO.38, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.

41.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment has at least two VH+VL domain combinations that are identical to each other, thereby binding to the same target protein; or that are different from each other, thereby binding to different target proteins. Preferably, each of the at least two VH+VL domain combinations comprises: (i) the amino acid sequence shown in SEQ ID NO. 31; the amino acid sequence shown in SEQ ID NO. 32; and the amino acid sequence shown in SEQ ID NO. 33; or (ii) the amino acid sequence shown in SEQ ID NO. 31; the amino acid sequence shown in SEQ ID NO. 32; and the amino acid sequence shown in SEQ ID NO. 41; More preferably, each of the at least two VH+VL domain combinations comprises: a light chain variable region containing the amino acid sequence shown in SEQ ID NO.11, SEQ ID NO.14 or SEQ ID NO.40, or a light chain variable region containing an amino acid sequence having at least 75% identity with the amino acid sequence; More preferably, the antibody or its antigen-binding fragment comprises: (1) The first VH+VL domain combination of B7-H3 (further, Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO.28, HCDR2 containing the amino acid sequence shown in SEQ ID NO.29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing SEQ ID NO.33; The second VH+VL domain combination of EGFR (further, the Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 34, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 35, and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 31, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 32, and LCDR3 containing SEQ ID NO. 33; or (1) The first VH+VL domain combination of B7-H3 (further, Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO.28, HCDR2 containing the amino acid sequence shown in SEQ ID NO.29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.31, LCDR2 containing the amino acid sequence shown in SEQ ID NO.32, and LCDR3 containing SEQ ID NO.41; The second VH+VL domain combination of EGFR (further, the Fab arm) comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 34, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 38, and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 31, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 32, and LCDR3 containing SEQ ID NO.

41.

7. The antibody or its antigen-binding fragment according to claim 5 or 6, characterized in that, The antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; Preferably, the antibody is a bispecific antibody.

8. An antibody-drug conjugate comprising the polypeptide of claim 1 or the antibody or antigen-binding fragment thereof of any one of claims 5 to 7, wherein the polypeptide or the antibody or antigen-binding fragment thereof is conjugated to a small molecule toxic compound via a linker.

9. A composition comprising the polypeptide of claim 1, the nucleic acid molecule of claim 2, the antibody or antigen-binding fragment thereof of any one of claims 5 to 7, or the antibody-drug conjugate of claim 8.

10. Use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the antibody or antigen-binding fragment thereof of any one of claims 5 to 7, the antibody-drug conjugate of claim 8, or the composition of claim 9 in the preparation of a medicament for treating or alleviating a disease.

11. An antibody or antigen-binding fragment thereof that binds to B7-H3, said antibody or antigen-binding fragment comprising heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3) as shown below: The HCDR1 comprising the amino acid sequence shown in SEQ ID NO.28, the HCDR2 comprising the amino acid sequence shown in SEQ ID NO.29, and the HCDR3 comprising the amino acid sequence shown in SEQ ID NO.30; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO.31, the LCDR2 comprising the amino acid sequence shown in SEQ ID NO.32, and the LCDR3 comprising the amino acid sequence shown in SEQ ID NO.

33. Preferably, the antibody binding to B7-H3 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise: (1) The amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with said amino acid sequence; or (2) The amino acid sequence shown in SEQ ID NO.13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

14.

12. An antibody or antigen-binding fragment thereof that binds to EGFR, said antibody or antigen-binding fragment comprising heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3) as shown below: The HCDR1 comprising the amino acid sequence shown in SEQ ID NO.34, the HCDR2 comprising the amino acid sequence shown in SEQ ID NO.35, and the HCDR3 comprising the amino acid sequence shown in SEQ ID NO.36; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO.29, the LCDR2 comprising the amino acid sequence shown in SEQ ID NO.30, and the LCDR3 comprising the amino acid sequence shown in SEQ ID NO.

31. Preferably, the antibody binding to EGFR or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise: (1) The amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence having at least 75% identity with said amino acid sequence; and, the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 75% identity with said amino acid sequence; or (2) The amino acid sequence shown in SEQ ID NO.15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

14.

13. Use of the antibody or antigen-binding fragment thereof as claimed in claim 11 or 12 in the preparation of a medicament for treating or alleviating a disease.

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