Anti-EGFR and / or PD-L1 antibody and application thereof

Abstract

The invention provides a polypeptide which can be used as a common light chain of a bispecific antibody to construct the bispecific antibody together with a heavy chain variable region or a heavy chain of an antibody with binding affinity and / or specificity to different target proteins. The invention also provides an anti-EGFR antibody, an anti-PD-L1 antibody and an EGFR * PDL1 bispecific antibody, wherein the antibodies comprise a common light chain variable region or a light chain.

Description

[0001] Cross-references to related applications

[0002] This patent application claims priority to Chinese invention patent application No. CN202410608740.7, filed on May 16, 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 against EGFR and / or PD-L1 comprising the peptide as a common light chain. Background Technology

[0004] In recent years, programmed death ligand 1 (PD-L1) inhibitors have demonstrated clinical benefits across a wide range of cancer types. However, despite these broad clinical benefits, only about 20% of patients receive immunotherapy containing these inhibitors. For example, in cancer treatment, the objective response rate with PD-1 / PD-L1 inhibitors alone is only 10%–30%, and is accompanied by a range of side effects.

[0005] Somatic activation mutations of the epidermal growth factor receptor (EGFR) are among the most common oncogenic drivers in cancers such as non-small cell lung cancer (NSCLC), metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer, with the highest prevalence in NSCLC. Molecularly targeted drugs targeting the EGFR signaling pathway have shown strong clinical efficacy; however, acquired resistance has been found to be inevitable in patients.

[0006] The EGFR×PD-L1 bispecific antibody is designed to simultaneously bind to the tumor-associated antigen, epidermal growth factor receptor (EGFR), and programmed death ligand 1 (PD-L1). In addition to its direct antitumor activity through EGFR inhibition, the EGFR×PD-L1 bispecific antibody can mediate effective antibody-dependent cytotoxicity (ADCC) and activate T-cell antitumor immunity by blocking the interaction between programmed death receptor 1 (PD-1) and its corresponding ligand PD-L1. Therefore, the EGFR×PD-L1 bispecific antibody can achieve enhanced therapeutic effects by directly inhibiting tumor growth and by blocking T-cell activation through tumor-targeted immune checkpoints.

[0007] 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, mismatch between the heavy and light chains may occur. Therefore, it has been proposed that a single polypeptide can be used as a universal light chain to pair with the heavy chain of any antibody that has binding affinity and specificity for a specific target protein (e.g., EGFR, PD-L1), thereby constructing an antibody (homodimer). Furthermore, this polypeptide can also be used as a universal light chain to pair with two or more antibody heavy chains, thus constructing bispecific or even multispecific antibodies (heterodimers, heteromultimers) with affinity and binding specificity for different targets. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a polypeptide, which, as a light chain variable region or light chain of an antibody, can be constructed into an antibody with a heavy chain variable region or heavy chain of an antibody having binding affinity and / or specificity to a specific target protein (antigen), or with two or more heavy chain variable regions or heavy chains of antibodies having 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 variable region or 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 whose amino acid sequence comprises: the amino acid sequence shown in SEQ ID NO.29; the amino acid sequence shown in SEQ ID NO.30; and the amino acid sequence shown in SEQ ID NO.31.

[0011] SEQ ID NO.29: SATSSIGYMH

[0012] SEQ ID NO.30: DTSKLAS

[0013] SEQ ID NO.31: QLWSSDPPYT

[0014] Further, the polypeptide comprises 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.15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.15.

[0015] SEQ ID NO.11:

[0016] QIVLTQSPAIMSASPGEKVTMTC SATSSIGYMHWYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTFYSLTISSMEAEDAATYYC QLWSSDPPYT FGGGTKLEIK

[0017] SEQ ID NO.15:

[0018] DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIK

[0019] The “at least 75% identity” as described in this invention covers 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.

[0020] Further, the polypeptide comprises the amino acid sequence shown in SEQ ID NO.38 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.22 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.22.

[0021] SEQ ID NO.38:

[0022] QIVLTQSPAIMSASPGEKVTMTC SATSSIGYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTFYSLTISSMEAEDAATYYC QLWSSDPPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0023] SEQ ID NO.22:

[0024] DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0025] The polypeptide provided by the present invention, as the light chain variable region or light chain of an antibody, can be used to construct antibodies by combining with the heavy chain variable region or heavy chain of antibodies that have binding affinity and / or specificity to different target proteins (antigens). The antibodies retain or have stronger binding affinity and / or specificity to the target protein (antigen) and corresponding biological activity.

[0026] 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 variable region or light chain of the antibody, can be combined with the heavy chain variable region or heavy chain of an antibody that has binding affinity and / or specificity to a certain target protein (antigen) to construct an antibody that still has binding affinity and / or specificity to that target protein (antigen) (a monospecific antibody). This antibody is a homodimer with two identical heavy chains and two identical light chains. Alternatively, for example, the polypeptide, as the light chain variable region or light chain of the antibody, can be combined with the heavy chain variable regions or heavy chains of two antibodies that have binding affinity and / or specificity to two target proteins (antigens) respectively to construct an antibody that still has binding affinity and / or specificity to each of the two target proteins (antigens) (a bispecific antibody). This antibody is a heterodimer with two different heavy chains and two identical light chains. Furthermore, the polypeptides provided by this invention can also be used to construct multispecific antibodies with binding affinity and / or specificity to more target proteins (antigens).

[0027] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide described in the first aspect.

[0028] 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.

[0029] 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.

[0030] According to a specific embodiment of the present invention, the target protein may be epidermal growth factor receptor (EGFR) or programmed death ligand 1 (PD-L1). In the context of the present invention, the epidermal growth factor receptor or programmed death ligand 1 may be mammalian, such as primate or rodent EGFR or PD-L1, more preferably human.

[0031] 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×PDL1 bispecific antibody provided by the present invention.

[0032] 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 a light chain or a light chain variable region; in other words, the polypeptide may serve as a light chain variable region or a light chain of the antibody or the antigen-binding fragment thereof.

[0033] 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 "antigen" are used interchangeably.

[0034] 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.

[0035] Therefore, for example, the antibody or antigen-binding fragment provided by the present invention contains the amino acid sequence provided by the first aspect of the present invention as its light chain CDRs in its light chain variable region; or, the antibody or antigen-binding fragment provided by the present invention contains the amino acid sequence shown in SEQ ID NO.11 or SEQ ID NO.15 or an amino acid sequence having at least 75% identity with said amino acid sequence as its light chain variable region (VL); or, the antibody or antigen-binding fragment provided by the present invention contains the amino acid sequence shown in SEQ ID NO.38 or SEQ ID NO.22 or an amino acid sequence having at least 75% identity with said amino acid sequence as its light chain.

[0036] Furthermore, the antibody or its antigen-binding fragment may further include a heavy chain variable region (VH), which, together with the light chain variable region, forms a binding domain targeting the target protein. According to a specific embodiment of the present invention, the target protein may be epidermal growth factor receptor (EGFR) or programmed cell death ligand 1 (PD-L1).

[0037] According to a specific embodiment of the present invention, the antibody or its antigen-binding fragment may comprise heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3) as shown below:

[0038] (1) HCDR1 containing the amino acid sequence shown in SEQ ID NO.26, HCDR2 containing the amino acid sequence shown in SEQ ID NO.27, HCDR3 containing the amino acid sequence shown in SEQ ID NO.28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31;

[0039] (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO.32, HCDR2 containing the amino acid sequence shown in SEQ ID NO.33, HCDR3 containing the amino acid sequence shown in SEQ ID NO.28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31;

[0040] (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 34, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 35, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31; or

[0041] (4) HCDR1 containing the amino acid sequence shown in SEQ ID NO.34, HCDR2 containing the amino acid sequence shown in SEQ ID NO.37, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31.

[0042] 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 may each comprise:

[0043] (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;

[0044] (2) 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. 12;

[0045] (3) 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. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11;

[0046] (4) 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. 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 15;

[0047] (5) The amino acid sequence shown in SEQ ID NO. 16 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. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0048] (6) The amino acid sequence shown in SEQ ID NO.17 or an amino acid sequence having at least 75% identity with 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.15.

[0049] The "at least 75% identity" of the present invention results in a maximum 25% difference between two amino acid sequences, which may exist in any frame region within the heavy chain variable region or the light chain variable region, or in any structural domain or sequence outside the heavy chain variable region and the light chain variable region.

[0050] The antibody or its antigen-binding fragment provided by this invention can be a mouse 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 this invention, the heavy chain constant region can be the heavy chain constant region of IgG1 or IgG4; the light chain constant region can 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.18 (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.19 (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.20 (hIgG1; Knob) or SEQ ID NO.21 (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.19 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0051] 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. 18; the light chain constant region contains the amino acid sequence shown in SEQ ID NO. 19. 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.

[0052] 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 contains 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 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. 29; LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30; and LCDR3 containing the amino acid sequence shown in SEQ ID NO. 31. Further, for example, each of the at least 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 or SEQ ID NO. 15, or an amino acid sequence having at least 75% identity with said amino acid sequence.

[0053] According to a specific embodiment of the present invention, the antibody or its antigen-binding fragment comprises:

[0054] (1) The first VH+VL domain combination of EGFR (further, the Fab arm), comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 26, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 27, and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 29, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 containing SEQ ID NO. 31; and

[0055] The second VH+VL domain combination of PD-L1 (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. 37, 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. 29, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 containing SEQ ID NO. 31; or

[0056] (2) The first VH+VL domain combination of EGFR (further, the Fab arm), comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 32; HCDR2 containing the amino acid sequence shown in SEQ ID NO. 33; and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 29; LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30; and LCDR3 containing the amino acid sequence shown in SEQ ID NO. 31; and

[0057] The second VH+VL domain combination of PD-L1 (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.37, 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.29; LCDR2 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31.

[0058] According to a specific embodiment of the present invention, the antibody or its antigen-binding fragment further comprises:

[0059] (1) The first VH+VL domain combination of EGFR (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. 14 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. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; and

[0060] The second VH+VL domain combination of PD-L1 (further, the Fab arm) comprises: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO. 17 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. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0061] (2) The first VH+VL domain combination of EGFR (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. 16 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. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; and

[0062] The second VH+VL domain combination of PD-L1 (further, the Fab arm) comprises: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO.17 or an amino acid sequence having at least 75% identity with 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.15.

[0063] In this case, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region. For example, the heavy 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 said amino acid sequence; the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity with said amino acid sequence. Alternatively, the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 20 (hIgG1; Knob) or SEQ ID NO. 21 (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 comprises the amino acid sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 75% identity with said amino acid sequence.

[0064] 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. 22 or an amino acid sequence having at least 75% identity with the amino acid sequence as the light chain.

[0065] According to specific embodiments of the present invention, the bispecific antibodies provided by the present invention are shown in Table 15 of the "Specific Embodiments" section. The bispecific antibodies in Table 15 include the following structural domains:

[0066] The bispecific antibody H22A12x58E3V4-hIgG1-new comprises two Fab arms targeting EGFR and PD-L1 respectively. One Fab arm contains a heavy chain variable region (SEQ ID NO. 14) and a light chain variable region (SEQ ID NO. 15) targeting EGFR, and the other Fab arm contains a heavy chain variable region (SEQ ID NO. 17) and a light chain variable region (SEQ ID NO. 15) targeting PD-L1. The bispecific antibody contains two identical light chain variable regions (SEQ ID NO. 15) and two corresponding identical light chains (SEQ ID NO. 22).

[0067] The bispecific antibody H103G9x58E3V4-hIgG1-new comprises two Fab arms targeting EGFR and PD-L1, respectively. One Fab arm contains a heavy chain variable region (SEQ ID NO.16) and a light chain variable region (SEQ ID NO.15) targeting EGFR, and the other Fab arm contains a heavy chain variable region (SEQ ID NO.17) and a light chain variable region (SEQ ID NO.15) targeting PD-L1. The bispecific antibody contains two identical light chain variable regions (SEQ ID NO.15) and two corresponding identical light chains (SEQ ID NO.22).

[0068] Furthermore, the bispecific antibodies in Table 15, each being an IgG type antibody, all contain two different heavy chains (heavy chain 1 and heavy chain 2) and the same two light chains:

[0069] The bispecific antibody H22A12x58E3V4-hIgG1-new 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.22 as the same light chain.

[0070] The bispecific antibody H103G9x58E3V4-hIgG1-new contains the amino acid sequence shown in SEQ ID NO.25 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.22 as the same light chain.

[0071] Fifthly, the present invention provides a composition comprising the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, or the antibody or antigen-binding fragment thereof described in the fourth aspect. The composition is preferably a pharmaceutical composition, which may further optionally comprise 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.

[0072] In a sixth 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, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament.

[0073] The drug may be an antibody-based drug used to treat diseases related to the expression of its target protein or antigen, such as EGFR and / or PD-L1 expression-related diseases. For example, the drug may be used to treat tumors or cancers such as lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), head and neck cancer, and colon cancer.

[0074] In a seventh aspect, the present invention provides a method of treating 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, or the pharmaceutical composition described in the fifth aspect of the present invention.

[0075] The method provided by this invention is used to treat tumors or cancer. Preferably, the tumor or cancer is lung cancer (such as non-small cell lung cancer, small cell lung cancer), head and neck cancer, or colon cancer. The subject is a mammal, preferably a primate or rodent, and more preferably a human.

[0076] Eighthly, 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:

[0077] (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 26, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 27, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31; or

[0078] (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO.32, HCDR2 containing the amino acid sequence shown in SEQ ID NO.33, HCDR3 containing the amino acid sequence shown in SEQ ID NO.28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31.

[0079] 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:

[0080] (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;

[0081] (2) 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. 12;

[0082] (3) 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, 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; or

[0083] (4) The amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence having at least 75% identity with 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.15.

[0084] The EGFR-binding antibody or its antigen-binding fragment 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.

[0085] 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 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.18 (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.19 (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.20 (hIgG1; Knob) or SEQ ID NO.21 (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.19 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0086] In a ninth aspect, the present invention also provides an antibody or antigen-binding fragment thereof that binds to PD-L1, 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] (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 34, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 35, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31; or

[0088] (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO.34, HCDR2 containing the amino acid sequence shown in SEQ ID NO.37, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31.

[0089] Preferably, the antibody binding to PD-L1 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:

[0090] (1) 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. 11 or an amino acid sequence having at least 75% identity with said amino acid sequence; or

[0091] (2) The amino acid sequence shown in SEQ ID NO.17 or an amino acid sequence having at least 75% identity with 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.15.

[0092] The antibody or antigen-binding fragment of PD-L1 provided by the present 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.

[0093] 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 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.18 (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.19 (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.20 (hIgG1; Knob) or SEQ ID NO.21 (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.19 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0094] In a tenth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof that binds to EGFR as described in the eighth aspect or the antibody or antigen-binding fragment thereof that binds to PD-L1 as described in the ninth aspect in the preparation of a medicament.

[0095] The drug may be an antibody-based drug used to treat diseases related to the expression of its target protein or antigen, such as EGFR and / or PD-L1 expression-related diseases. For example, the drug may be used to treat tumors or cancers, such as, preferably, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), head and neck cancer, or colon cancer.

[0096] This invention provides a polypeptide, which, experimentally demonstrated, can serve as a common light chain or light chain variable region of an antibody, combined with the heavy chain or heavy chain variable region of an antibody possessing binding affinity and / or specificity to other target proteins (antigens), to construct a bispecific antibody. This antibody retains or even exhibits binding affinity and biological activity against two different target proteins (antigens). 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 antibodies. Furthermore, this invention also provides antibodies comprising the polypeptide as a common light chain variable region or light chain, such as anti-EGFR antibodies, anti-PD-L1 antibodies, and EGFR×PD-L1 bispecific antibodies. Attached Figure Description

[0097] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0098] Figure 1 The binding activity of the murine anti-PD-L1 antibody and control antibody to PD-L1 overexpressing cells was demonstrated;

[0099] Figure 2 The blocking activity of murine anti-PD-L1 antibody and control antibody against PD-L1 binding to PD-1 was demonstrated;

[0100] Figure 3 The blocking activity of the murine anti-PD-L1 antibody and the control antibody against PD-L1 / PD-1 signaling was demonstrated.

[0101] Figure 4 The affinity of the murine anti-PD-L1 antibody for human PD-L1 was demonstrated;

[0102] Figure 5 The binding activity of the humanized anti-PD-L1 antibody and control antibody to PD-L1 overexpressing cells was demonstrated;

[0103] Figure 6 The humanized antibody against PD-L1 and the control antibody demonstrated their blocking activity against the binding of PD-L1 to PD-1;

[0104] Figure 7 The blocking activity of humanized anti-PD-L1 antibodies and control antibodies against PD-L1 / PD-1 signaling was demonstrated;

[0105] Figure 8 The binding activity of the anti-EGFR murine antibody and control antibody to EGFR-overexpressing cells was demonstrated;

[0106] Figure 9 The blocking activity of the murine anti-EGFR antibody and the control antibody against the binding of EGFR to EGF was demonstrated.

[0107] Figure 10 The blocking activity of the murine anti-EGFR antibody and the control antibody against EGFR signaling was demonstrated.

[0108] Figure 11 The affinity of the murine anti-EGFR antibody for EGFR was demonstrated;

[0109] Figure 12 The binding activity of the humanized anti-EGFR antibody and control antibody to EGFR-overexpressing cells was demonstrated;

[0110] Figure 13 The humanized anti-EGFR antibody and control antibody were shown to block the binding of EGFR to EGF.

[0111] Figure 14The blocking activity of the humanized anti-EGFR antibody and the control antibody against EGFR signaling was demonstrated;

[0112] Figure 15 The binding activity of the EGFR×PDL1 bispecific antibody and control antibody to PD-L1 overexpressing cells was demonstrated.

[0113] Figure 16 The blocking activity of the EGFR×PDL1 bispecific antibody and the control antibody against the binding of PD-L1 to PD-1 was demonstrated.

[0114] Figure 17 The blocking activity of the EGFR×PDL1 bispecific antibody and the control antibody against PD-L1 / PD-1 signaling was demonstrated.

[0115] Figure 18 The binding activity of the EGFR×PDL1 bispecific antibody and control antibody to EGFR-overexpressing cells was demonstrated.

[0116] Figure 19 The blocking activity of the EGFR×PDL1 bispecific antibody and the control antibody against the binding of EGFR to EGF was demonstrated.

[0117] Figure 20 The blocking activity of the EGFR×PDL1 bispecific antibody and control antibody against EGFR signaling was demonstrated.

[0118] Figure 21 The killing activity of the EGFR×PDL1 bispecific antibody and control antibody against tumor cells was demonstrated. Detailed Implementation

[0119] 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.

[0120] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0121] The following constant region sequence was used when constructing the antibody:

[0122] Human heavy chain CH1 and FC sequences (SEQ ID NO.18):

[0123] hIgG1-WT:

[0124] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0125] > Human light chain CL sequence (SEQ ID NO.19):

[0126] hkappa:

[0127] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0128] > Human heavy chain Knob CH1 and FC sequence (hIgG1; SEQ ID NO.20):

[0129] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0130] Human heavy chain Hole CH1 and FC sequences (hIgG1; SEQ ID NO.21):

[0131] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0132] The following control antibodies were used in the following examples:

[0133] Atezolizumab: Self-made by the applicant (Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., "Kangyuan Bochuang"), Cat#: KA-1303-02;

[0134] Cetuximab: Self-made by the applicant, Cat#: KA-1398-01.

[0135] Unless otherwise specified, the target proteins EGFR and PD-L1 involved in the examples are human proteins.

[0136] Example 1 Screening of common light chains

[0137] 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.

[0138] KV4-59 (SEQ ID NO.1):

[0139] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPP FGSGTKLEIK

[0140] The above sequence was modified. Since CDR3 of the light chain (see underlined; obtained according to the KABAT definition) plays an important role in the affinity between the antibody and antigen, an amino acid was added after CDR3 to improve the affinity for potential antigens. The following three sequences were obtained:

[0141] KV4-59-1 (SEQ ID NO.2):

[0142] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPYT FGSGTKLEIK

[0143] KV4-59-2 (SEQ ID NO.3):

[0144] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPWT FGSGTKLEIK

[0145] KV4-59-3 (SEQ ID NO.4):

[0146] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPFT FGSGTKLEIK

[0147] 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.

[0148] The heavy chain variable region sequence of mouse anti-chicken egg lysozyme antibody (SEQ ID NO.5):

[0149] LDVKLQESGPGLVKPSQSLSLTCSVTGYSFTSGYYWNWIRLFPGNKLEWMGYVSYFGTNNYNPSLKNRFSITRDTSKNQFFLKLNSVSTEDTATYYCARGEEWDWSFDVWGTGTTVTVSS

[0150] 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.

[0151] Example 2 Construction of transgenic mice

[0152] 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.

[0153] 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.

[0154] 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.

[0155] Example 3 Preparation of hybridoma cells secreting anti-EGFR and anti-PD-L1 antibodies

[0156] Mice constructed in Example 2 were immunized using EGFR-mFc (Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. (“Kangyuan Bochuang”), Cat#: KP-1151) 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.

[0157] A murine antibody against EGFR was obtained from a positive hybridoma cell line. 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): 22A12-2 (murine antibody against EGFR 1; also named "KD-22-0198")

[0158] >EGFR-22A12-2, VH (SEQ ID NO.10; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.26 / SEQ IDNO.27 / SEQ ID NO.28):

[0159] EVQLQQSGPELVKPGASVKISCKASGYTFT DYYMN WVKQSHGKSLEWIG DINPNDGGTSYNQKFKG KATLTVDKSSSTAYMELRRSLTSEDSAVYYCAR EILYYVTSFYFDY WGQGTTLTVSS

[0160] >EGFR-22A12-2, VL (SEQ ID NO.11; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ IDNO.30 / SEQ ID NO.31):

[0161] QIVLTQSPAIMSASPGEKVTMTC SATSSIGYMH WYQQKSGTSPKRWIY DTSKLASGVPARFSGSGSGTFYSLTISSMEAEDAATYYC QLWSSDPPYT FGGGTKLEIK

[0162] 103G9-3 (anti-EGFR murine antibody 2; also named "KD-22-0220")

[0163] >EGFR-103G9-3, VH (SEQ ID NO.12; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.32 / SEQ IDNO.33 / SEQ ID NO.28):

[0164] EVQLQQSGPELVKPGASVKISCKASGYTFT DYYIN WVKQSHGKSLEWIG DINPNNDDTNYNQKFKG KATLTVDKSSSTAYMELRRSLTSEDSAVYYCAR EILYYVTSFYFDY WGQGTTLTVSS

[0165] >EGFR-103G9-3, VL (SEQ ID NO.11; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ IDNO.30 / SEQ ID NO.31):

[0166] QIVLTQSPAIMSASPGEKVTMTC SATSSIGYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTFYSLTISSMEAEDAATYYC QLWSSDPPYT FGGGTKLEIK

[0167] Mice constructed in Example 2 were immunized using PDL1-mFc (Kangyuan Bochuang, Cat#: KP-1008) 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 PDL1-His (Kangyuan Bochuang, Cat#: KP-1002) was detected using ELISA, yielding multiple positive hybridoma cell lines secreting anti-PD-L1 antibodies.

[0168] Anti-PD-L1 murine antibodies were obtained from positive hybridoma cell lines, and their variable region sequences are shown below (heavy chain and light chain CDRs are underlined):

[0169] 58E3-2 (Mouse antibody against PD-L1; also named "KD-22-0227")

[0170] >PDL1-58E3-2, VH (SEQ ID NO.13; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.34 / SEQ IDNO.35 / SEQ ID NO.36):

[0171] QVQLKESGGGLVKPGGSLKLSCAASGFTFS DYGIH WVRQAPEKGLEWVA FISSGSSTIYYADTVKG RFTISRDNAKNTLFLQMTSLRSEDTAMYYCTR RDFYGSADAMDY WGQGTSVTVSS

[0172] >PDL1-58E3-2, VL (SEQ ID NO.11; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ IDNO.30 / SEQ ID NO.31):

[0173] QIVLTQSPAIMSASPGEKVTMTC SATSSIGYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTFYSLTISSMEAEDAATYYC QLWSSDPPYT FGGGTKLEIK

[0174] Anti-EGFR murine antibodies 22A12-2 and 103G9-3, and anti-PD-L1 murine antibody 58E3-2 have the same light chain variable region.

[0175] Example 4 Construction of humanized and chimeric antibodies against EGFR and PD-L1

[0176] The heavy chain and light chain variable regions of two anti-EGFR murine antibodies were humanized to obtain humanized antibody sequences. The coding sequences of these humanized antibody sequences were then linked to the coding sequences of the heavy chain constant region (SEQ ID NO. 18) of human IgG1 and the light chain constant region (SEQ ID NO. 19) of human kappa, respectively. The resulting coding genes were cloned into a eukaryotic expression vector, expressed, and yielded two humanized anti-EGFR antibodies, named H22A12-2V1-hIgG1 and H103G9-3V2-hIgG1, respectively. Their heavy chain and light chain variable region sequences and full-length heavy and light chains are shown below (where the heavy chain and light chain CDRs are underlined).

[0177] In addition, the coding sequences of the heavy chain variable region and light chain variable region of two anti-EGFR murine antibodies were linked to the coding sequences of the heavy chain constant region (SEQ ID NO.18) of human IgG1 and the light chain constant region (SEQ ID NO.19) of human kappa, respectively. The resulting coding genes were cloned into a eukaryotic expression vector, expressed, and used to obtain anti-EGFR chimeric antibodies.

[0178] H22A12-2V1-hIgG1 (humanized anti-EGFR antibody 1; also named "KA-1063")

[0179] >EGFR-H22A12-2V1-hIgG1, VH (SEQ ID NO.14; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.26 / SEQ ID NO.27 / SEQ ID NO.28):

[0180] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYYMN WVRQAPGQGLEWIG DINPNDGGTSYNQ

[0181] KFKG RATLTVDKSTSTAYMELSSLRSEDTAVYYCAR EILYYVTSFYFDY WGQGTTVTVSS

[0182] >EGFR-H22A12-2V1-hIgG1, VL (SEQ ID NO.15; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31):

[0183] DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIK

[0184] H103G9-3V2-hIgG1 (humanized anti-EGFR antibody 2; also named "KA-1083")

[0185] >EGFR-H103G9-3V2-hIgG1, VH (SEQ ID NO.16; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.32 / SEQ ID NO.33 / SEQ ID NO.28):

[0186] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYYIN WVRQAPGQGLEWIG DINPNNDDTNYNQKFKG RATLTVDKSISTAYMELSRLRSDDTAVYYCAR EILYYVTSFYFDY WGQGTTVTVSS

[0187] >EGFR-H103G9-3V2-hIgG1, VL (SEQ ID NO.15; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31):

[0188] DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIK

[0189] The heavy chain and light chain variable regions of a murine anti-PD-L1 antibody were humanized to obtain a humanized antibody sequence. The coding sequences of the humanized antibody sequence were then linked to the coding sequences of the heavy chain constant region (SEQ ID NO.18) of human IgG1 and the light chain constant region (SEQ ID NO.19) of human kappa, respectively. The resulting coding genes were cloned into a eukaryotic expression vector, expressed, and a humanized anti-PD-L1 antibody was obtained, named H58E3-2V4-2-hIgG1. Its heavy chain and light chain variable region sequences and full-length heavy and light chains are shown below (where the heavy chain and light chain CDRs are underlined).

[0190] In addition, the coding sequences of the heavy chain variable region and light chain variable region of a murine anti-PD-L1 antibody were linked to the coding sequences of the heavy chain constant region (SEQ ID NO.18) of human IgG1 and the light chain constant region (SEQ ID NO.19) of human kappa, respectively. The resulting coding genes were cloned into a eukaryotic expression vector, expressed, and a chimeric anti-PD-L1 antibody was obtained.

[0191] H58E3-2V4-2-hIgG1 (humanized antibody against PD-L1; also named "KA-2016")

[0192] >PDL1-H58E3-2V4-2-hIgG1, VH (SEQ ID NO.17; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.34 / SEQ ID NO.37 / SEQ ID NO.36)

[0193] QVQLVESGGGLVKPGGSLRLSCAASGFTFS DYGIH WIRQAPGKGLEWVA FISSGSSTIYYADAVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCTR RDFYGSADAMDY WGQGTTVTVSS

[0194] >PDL1-H58E3-2V4-2-hIgG1, VL (SEQ ID NO.15; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31)

[0195] DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIK

[0196] The anti-EGFR humanized antibodies H22A12-2V1-hIgG1 and H103G9-3V2-hIgG1, and the anti-PD-L1 humanized antibody H58E3-2V4-2-hIgG1 have the same light chain variable region (SEQ ID NO.15) and the same light chain (SEQ ID NO.22).

[0197] SEQ ID NO.22 (VL: SEQ ID NO.15; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ IDNO.30 / SEQ ID NO.31; LC: SEQ ID NO.19)

[0198] DIQLTQSPSTLSASVGDRVTITC SATSSIGYMH WYQQKPGTSPKRWIY DTSKLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QLWSSDPPYT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0199] Two anti-EGFR chimeric antibodies and one anti-PD-L1 chimeric antibody have the same light chain variable region (SEQ ID NO. 14) and the same light chain (SEQ ID NO. 38).

[0200] SEQ ID NO.38 (VL: SEQ ID NO.11; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ IDNO.30 / SEQ ID NO.31; LC: SEQ ID NO.19)

[0201] QIVLTQSPAIMSASPGEKVTMTC SATSSIGYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTFYSLTISSMEAEDAATYYC QLWSSDPPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0202] Example 5 Binding activity of murine anti-PD-L1 antibody to PD-L1 overexpressing cells

[0203] The antibodies to be tested were serially diluted and co-incubated with PD-L1-overexpressing 293T cells (Kangyuan Bochuang, Cat#: KC-0205), followed by co-incubation with the anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307) or the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect antibody signals bound to the cells. The results are shown in Table 1 and [Table data missing]. Figure 1 .

[0204] Table 1. Binding activities of murine anti-PD-L1 antibody and control antibody with PD-L1 overexpressing cells

[0205] Antibody EC50 (μg / mL) Top Atezolizumab 0.2443 370.9 58E3-2 0.2452 354.5

[0206] Example 6 The blocking activity of murine anti-PD-L1 antibodies against PD-L1 binding to PD-1

[0207] The antibodies to be tested were serially diluted and co-incubated with 2 μg / ml of PDL1-mFc (Kangyuan Biotech, Cat#: KP-1008) or PDL1-hFc (Kangyuan Biotech, Cat#: KP-1009), then co-incubated with PD-1 overexpressing 293T cells (Kangyuan Biotech, Cat#: KC-0204), and finally co-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). Flow cytometry was used to detect the PDL1-Fc signal bound to the cells. The results are shown in Table 2. Figure 2 .

[0208] Table 2. Blocking activity of murine anti-PD-L1 antibodies and control antibodies against PD-L1 binding to PD-1

[0209] Antibody IC50 (μg / mL) Atezolizumab 2.184 58E3-2 0.9857

[0210] Example 7 The blocking activity of murine anti-PD-L1 antibodies against PD-L1 / PD-1 signaling.

[0211] The antibodies to be tested were serially diluted and co-incubated with Jurkat NFAT-PD1-luc cells (Kangyuan Bochuang, Cat#: KC-1503, expressing luciferase after NFAT signal activation) and 293T OS8-hPDL1 cells (Kangyuan Bochuang, Cat#: KC-1148, expressing OKT3 antibody on the cell surface). The activation of luciferase signal was detected; cells without antibody incubation were considered to have basal activation. A correlation curve was plotted between the fold increase in activation intensity after antibody addition and the antibody concentration, and the EC50 of the antibody was calculated. The results are shown in Table 3. Figure 3 .

[0212] Table 3. Blocking activity of murine anti-PD-L1 antibodies and control antibodies against PD-L1 / PD-1 signaling.

[0213] Antibody EC50 (μg / mL) Top Atezolizumab 0.900 3.314 58E3-2 1.001 3.518

[0214] Example 8 Affinity of murine anti-PD-L1 antibodies to PD-L1

[0215] ForteBio Octet assay was used to detect the affinity of the antibody for PD-L1. The mobile phase hPDL1-His (Kangyuan Bochuang, Cat#: KP-1002) concentrations were 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM, respectively. The affinity assay results are shown in Table 4. Figure 4 .

[0216] Table 4. Affinity of murine anti-PD-L1 antibodies and control antibodies to human PD-L1

[0217] Antibody KD(M) kon(1 / Ms) kdis(1 / s) 58E3-2 4.31E-10 1.43E+06 6.18E-04

[0218] Example 9 Binding activity of humanized anti-PD-L1 antibodies to PD-L1 overexpressing cells

[0219] The antibodies to be tested were serially diluted and co-incubated with PD-L1-overexpressing 293T cells (Kangyuan Biotech, Cat#: KC-0205), followed by co-incubation with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect antibody signals bound to the cells; the results are shown in Table 5. Figure 5 .

[0220] Table 5. Binding activity of humanized anti-PD-L1 antibodies and control antibodies to PD-L1 overexpressing cells

[0221] Antibody EC50 (μg / mL) MFI.Max Atezolizumab 0.05279 448000 H58E3-2V4-2-hIgG1 0.04187 474000

[0222] Example 10 The blocking activity of humanized anti-PD-L1 antibodies against PD-L1 binding to PD-1

[0223] The antibody to be tested was serially diluted and co-incubated with 1.2 μg / ml PDL1-mFc (Kangyuan Bochuang, Cat#: KP-1008), then co-incubated with PD-1 overexpressing 293T cells (Kangyuan Bochuang, Cat#: KC-0204), 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 PDL1-mFc signal bound to the cells. The results are shown in Table 6. Figure 6 .

[0224] Table 6. Blocking activity of humanized anti-PD-L1 antibodies and control antibodies against PD-L1 binding to PD-1

[0225] Antibody IC50 (μg / mL) Atezolizumab 0.0305 H58E3-2V4-2-hIgG1 0.02289

[0226] Example 11 The blocking activity of humanized anti-PD-L1 antibodies against PD-L1 / PD-1 signaling

[0227] The antibodies to be tested were serially diluted and co-incubated with Jurkat NFAT-PD1-luc cells (Kangyuan Bochuang, Cat#: KC-1503, expressing luciferase after NFAT signal activation) and 293T OS8-hPDL1 cells (Kangyuan Bochuang, Cat#: KC-1148, expressing OKT3 antibody on the cell surface). The activation signal of luciferase was detected; cells without antibody incubation were considered to have basal activation. A correlation curve was plotted between the fold increase in activation intensity after antibody addition and the antibody concentration, and the EC50 of the antibody was calculated. The results are shown in Table 7. Figure 7 .

[0228] Table 7. Blocking activity of humanized anti-PD-L1 antibodies and control antibodies against PD-L1 / PD-1 signaling.

[0229] Antibody EC50 (μg / ml) Top Atezolizumab 0.098 5.944 H58E3-2V4-2-hIgG1 0.081 6.169

[0230] Example 12 Binding activity of anti-EGFR murine antibodies to EGFR-overexpressing cells

[0231] The antibodies to be tested were serially diluted and co-incubated with EGFR-overexpressing CT26 cells (Kangyuan Bochuang, Cat#: KC-1451), followed by co-incubation with the anti-mouse secondary antibody PE anti-mouse IgG Fc (Biolegend, Cat#: 405307) or the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect antibody signals bound to the cells. The results are shown in Table 8. Figure 8 .

[0232] Table 8. Binding activities of anti-EGFR murine antibodies and control antibodies to EGFR-overexpressing cells.

[0233] Antibody EC50 (μg / mL) Cetuximab 0.01554 22A12-2 0.02418 103G9-3 0.02256

[0234] Example 13 The blocking activity of murine anti-EGFR antibodies against EGFR-EGF binding

[0235] 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 using a microplate reader, and the IC50 of the antibody was calculated. The results are shown in Table 9. Figure 9 .

[0236] Table 9. Blocking activity of murine anti-EGFR antibodies and control antibodies against EGFR-EGF binding.

[0237]

[0238]

[0239] Example 14 The blocking activity of murine anti-EGFR antibodies against EGFR signaling

[0240] The antibody to be tested was serially diluted and co-incubated with 293T-NFAT-Luc2-EGFR cells (Kangyuan Bochuang, Cat#: KC-2952, expressing luciferase after EGFR signal activation) for 30 minutes. Then, EGF-His (20 ng / ml, Acro, Cat#: EGF-H52H3) was added and the cells were cultured overnight. The activated luciferase signal was detected. Cells without EGF incubation were considered to have basal activation. A correlation curve was plotted between the fold increase in activation intensity after EGF addition and the antibody concentration, and the IC50 of the antibody was calculated. The results are shown in Table 10. Figure 10 .

[0241] Table 10. Blocking activity of murine anti-EGFR antibodies and control antibodies against EGFR signaling

[0242] Antibody IC50 (μg / mL) Cetuximab 0.3462 22A12-2 0.3735 103G9-3 0.2595

[0243] Example 15 Affinity of murine anti-EGFR antibodies to EGFR

[0244] ForteBio Octet assay was used to detect the antibody affinity for EGFR. The mobile phase EGFR-His (Kangyuan Bochuang, Cat#: KP-1150) concentrations were 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM, respectively. Affinity test results are shown in Table 11. Figure 11(22A12-2).

[0245] Table 11. Affinity of murine anti-EGFR antibodies and control antibodies to EGFR

[0246] Antibody KD(M) kon(1 / Ms) kdis(1 / s) 22A12-2 1.15E-09 9.65E+05 1.11E-03 103G9-3 7.31E-10 9.89E+05 7.23E-04

[0247] Example 16 Binding activity of humanized anti-EGFR antibodies to EGFR-overexpressing cells

[0248] The antibodies to be tested were serially diluted and co-incubated with EGFR-overexpressing CT26 cells (Kangyuan Biotech, Cat#: KC-1451), followed by co-incubation with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect antibody signals bound to the cells; the results are shown in Table 12. Figure 12 .

[0249] Table 12. Binding activities of humanized anti-EGFR antibodies and control antibodies with EGFR-overexpressing cells

[0250]

[0251]

[0252] Example 17 Humanized anti-EGFR antibodies' blocking activity against EGFR-EGF binding

[0253] The antibody to be tested was serially diluted and co-incubated with 0.4 μg / ml EGFR-mFc (Kangyuan Bochuang, Cat#: KP-1148), then incubated with an ELISA plate coated with EGF-His (2 μg / ml, Acro, Cat#: EGF-H52H3), and finally co-incubated with the anti-mouse secondary antibody HRP anti-mouse IgG (SIGMA, Cat#: A9309). The binding signal was detected using a microplate reader. The results are shown in Table 13. Figure 13 .

[0254] Table 13. Blocking activity of humanized anti-EGFR antibodies and control antibodies against EGFR-EGF binding.

[0255] Antibody IC50 (μg / mL) Cetuximab 0.01195 H22A12-2V1-hIgG1 0.03566 H103G9-3V2-hIgG1 0.01686

[0256] Example 18 The blocking activity of humanized anti-EGFR antibodies against EGFR signaling

[0257] The antibody to be tested was serially diluted and co-incubated with 293T-NFAT-Luc2-EGFR (Kangyuan Bochuang, Cat#: KC-2952, expressing luciferase after EGFR signal activation) for 30 minutes. Then, EGF-His (20 ng / ml, Acro, Cat#: EGF-H52H3) was added and the mixture was incubated overnight. The activated luciferase signal was detected; basal activation was indicated without EGF incubation. A correlation curve was plotted between the fold increase in activation intensity after EGF addition and the antibody concentration, and the IC50 of the antibody was calculated. The results are shown in Table 14. Figure 14 .

[0258] Table 14. Blocking activity of humanized anti-EGFR antibodies and control antibodies against EGFR signaling

[0259] Antibody IC50 (μg / ml) Cetuximab 0.2793 H22A12-2V1-hIgG1 0.3891 H103G9-3V2-hIgG1 0.4592

[0260] Example 19 Construction of EGFR×PD-L1 bispecific antibody

[0261] By linking the common light chain variable region (SEQ ID NO.15) with the coding sequence of the human kappa light chain constant region (SEQ ID NO.19), the common light chain of the bispecific antibody (SEQ ID NO.22) is obtained.

[0262] The heavy chain variable region (SEQ ID NO. 14) of the anti-EGFR humanized antibody 1, namely H22A12-2V1-hIgG1, was linked to the coding sequence of the human heavy chain Knob CH1 and FC sequence (SEQ ID NO. 20). In addition, the heavy chain variable region (SEQ ID NO. 16) of the anti-EGFR humanized antibody 2, namely H103G9-3V2-hIgG1, was linked to the coding sequence of the human heavy chain Knob CH1 and FC sequence (SEQ ID NO. 20). The heavy chain variable region (SEQ ID NO. 17) of the anti-PD-L1 humanized antibody, namely H58E3-2V4-2-hIgG1, was linked to the coding sequence of the human heavy chain Hole CH1 and FC sequence (SEQ ID NO. 21).

[0263] The obtained coding sequences were cloned into eukaryotic expression vectors to obtain four recombinant expression plasmids (one plasmid encoding a common light chain, two plasmids encoding heavy chains targeting EGFR, and one plasmid encoding heavy chains targeting PD-L1). Transient co-transfection of three plasmids (selecting one of the two plasmids encoding the heavy chain targeting EGFR) using the HEK293F expression system was employed to produce EGFR×PD-L1 bispecific antibodies. In short, for a 1L shake flask, HEK293F cells were used at a rate of 1×10⁻⁶ cells / mL. 6Inoculate 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.

[0264] The domains and their sequences contained in the obtained EGFR×PD-L1 bispecific antibody are shown in Table 15 and below (the italicized parts in the sequences below are constant regions).

[0265] Table 15. EGFR×PD-L1 bispecific antibodies containing a common light chain

[0266]

[0267] H22A12x58E3V4-hIgG1-new (EGFR×PD-L1 bispecific antibody 1; also named "KA-2888")>EGFR-22A12-2-Knob (heavy chain 1, SEQ ID NO.23; VH: SEQ ID NO.14; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.26 / SEQ ID NO.27 / SEQ ID NO.28; CH[Knob]: SEQ ID NO.20)

[0268]

[0269] >PDL1-58E3-2-Hole (Heavy chain 2, SEQ ID NO.24; VH: SEQ ID NO.17; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.34 / SEQ ID NO.37 / SEQ ID NO.36; CH[Hole]: SEQ ID NO.21)

[0270]

[0271] >Common light chain (SEQ ID NO.22; VL: SEQ ID NO.15; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31; LC: SEQ ID NO.19)

[0272]

[0273] H103G9x58E3V4-hIgG1-new (EGFR×PD-L1 bispecific antibody 2; also named "KA-2887")>EGFR-H103G9-3V2-Knob (heavy chain 1, SEQ ID NO.25; VH: SEQ ID NO.16; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.32 / SEQ ID NO.33 / SEQ ID NO.28; CH[Knob]: SEQ ID NO.20)

[0274]

[0275] PDL1-58E3-2-Hole (Heavy chain 2, SEQ ID NO.24; VH: SEQ ID NO.17; HCDR1 / HCDR2 / HCDR3: SEQ ID NO.34 / SEQ ID NO.37 / SEQ ID NO.36; CH [Hole]: SEQ ID NO.21)

[0276]

[0277] >Common light chain (SEQ ID NO.22; VL: SEQ ID NO.15; LCDR1 / LCDR2 / LCDR3: SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31; LC: SEQ ID NO.19)

[0278]

[0279] Example 20 Binding activity of EGFR×PDL1 bispecific antibody to PD-L1 overexpressing cells

[0280] The antibody to be tested was serially diluted and co-incubated with PD-L1-overexpressing 293T cells (Kangyuan Biotech, Cat#: KC-0205), and then co-incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect the antibody signal bound to the cells. The results are shown in Table 16. Figure 15 .

[0281] Table 16. Binding activities of EGFR×PDL1 bispecific antibody and control antibody with PD-L1 overexpressing cells

[0282] Antibody EC50 (μg / mL) MFI.Max Atezolizumab 0.05471 881000 H103G9x58E3V4-hIgG1-new 0.05338 931000 H22A12x58E3V4-hIgG1-new 0.0432 935000

[0283] Example 21 EGFR×PDL1 bispecific antibody blocking activity against PD-L1 and PD-1 binding

[0284] The antibody to be tested was serially diluted and co-incubated with 1.2 μg / ml PDL1-mFc (Kangyuan Bochuang, Cat#: KP-1008), then co-incubated with PD-1 overexpressing 293T cells (Kangyuan Bochuang, Cat#: KC-0204), 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 PDL1-mFc signal bound to the cells. The results are shown in Table 17. Figure 16 .

[0285] Table 17. Blocking activity of EGFR×PDL1 bispecific antibody and control antibody against PD-L1-PD-1 binding

[0286] Antibody IC50 (μg / mL) Atezolizumab 0.03796 H103G9x58E3V4-hIgG1-new 0.1255 H22A12x58E3V4-hIgG1-new 0.1218

[0287] Example 22 Blocking activity of EGFR×PDL1 bispecific antibody against PD-L1 / PD-1 signaling

[0288] The antibodies to be tested were serially diluted and co-incubated with Jurkat NFAT-PD1-luc cells (Kangyuan Bochuang, Cat#: KC-1503, expressing luciferase after NFAT signal activation) and 293T OS8-hPDL1 cells (Kangyuan Bochuang, Cat#: KC-1148, expressing OKT3 antibody on the cell surface). The activation of luciferase signal was detected; cells without antibody incubation were considered to have basal activation. A correlation curve was plotted between the fold increase in activation intensity after antibody addition and the antibody concentration, and the EC50 of the antibody was calculated. The results are shown in Table 18. Figure 17 .

[0289] Table 18. Blocking activity of EGFR×PDL1 bispecific antibody and control antibody against PD-L1 / PD-1 signaling

[0290]

[0291]

[0292] Example 23 Binding activity of EGFR×PDL1 bispecific antibody to EGFR-overexpressing cells

[0293] The antibodies to be tested were serially diluted and co-incubated with EGFR-overexpressing CT26 cells (Kangyuan Biotech, Cat#: KC-1451), followed by co-incubation with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat#: 2010-09). Flow cytometry was used to detect antibody signals bound to the cells; the results are shown in Table 19. Figure 18 .

[0294] Table 19. Binding activities of EGFR×PDL1 bispecific antibody and control antibody with EGFR-overexpressing cells

[0295] Antibody EC50 (μg / mL) MFI.Max Cetuximab 0.007066 83402 H103G9x58E3V4-hIgG1-new 0.06363 113452 H22A12x58E3V4-hIgG1-new 0.06942 166429

[0296] Example 24 EGFR×PDL1 bispecific antibody blocking activity against EGFR-EGF binding

[0297] The antibody to be tested was serially diluted and co-incubated with EGFR-overexpressing CT26 cells (Kangyuan Bochuang, Cat#: KC-1451), 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. The results are shown in Table 20. Figure 19 .

[0298] Table 20. Blocking activity of EGFR×PDL1 bispecific antibody and control antibody against EGFR-EGF binding

[0299] Antibody IC50 (μg / mL) Cetuximab 0.0014 <![CDATA[ H103G9x58E3V4-hIgG1-new ]]> 0.023 H22A12x58E3V4-hIgG1-new 0.020

[0300] Example 25 Blocking activity of EGFR×PDL1 bispecific antibody against EGFR signaling

[0301] The antibody to be tested was serially diluted and co-incubated with 293T-NFAT-Luc2-EGFR cells (Kangyuan Bochuang, Cat#: KC-2952, expressing luciferase after EGFR signal activation) for 30 minutes. Then, EGF-His (20 ng / ml, Acro, Cat#: EGF-H52H3) was added and the cells were cultured overnight. Activation of luciferase signal was detected. Cells without EGF incubation were considered to have basal activation. The fold increase in activation intensity after EGF addition was plotted against antibody concentration, and the results are shown in Table 21. Figure 20 .

[0302] Table 21. Blocking activity of EGFR×PDL1 bispecific antibody and control antibody against EGFR signaling

[0303] Antibody IC50 (μg / ml) Cetuximab 0.152 H103G9x58E3V4-hIgG1-new 1.098 H22A12x58E3V4-hIgG1-new 1.615

[0304] Example 26 Detection of the affinity of EGFR×PDL1 bispecific antibody for human EGFR and PD-L1

[0305] The ForteBio Octet assay was used to detect the affinity of the antibody for EGFR and PD-L1. The mobile phase consisted of human EGFR-His (Kangyuan Bochuang, Cat#: KP-1150) and human PDL1-His (Kangyuan Bochuang, Cat#: KP-1002), diluted sequentially to 200, 100, 50, 25, 12.5, 6.25, and 3.12 nM. The results are shown in Table 22.

[0306] Table 22. Affinity of EGFR×PD-L1 bispecific antibody and control antibody to EGFR and PD-L1

[0307] Antibody antigen ka(1 / Ms) kd(1 / s) KD(M) Atezolizumab hPDL1-His 4.21E+05 3.31E-04 7.85E-10 H22A12x58E3V4-hIgG1-new hPDL1-His 1.31E+06 8.70E-04 6.62E-10 H103G9x58E3V4-hIgG1-new hPDL1-His 1.35E+06 8.10E-04 6.02E-10 Cetuximab hEGFR-His 9.93E+05 1.77E-03 1.78E-09 H22A12x58E3V4-hIgG1-new hEGFR-His 1.26E+06 3.51E-03 2.79E-09 H103G9x58E3V4-hIgG1-new hEGFR-His 1.27E+06 4.71E-03 3.71E-09

[0308] Example 27 Detection of the cytotoxic activity of EGFR×PDL1 bispecific antibody against tumor cells

[0309] Lung cancer cells NCI-H292 (Kangyuan Bochuang, Cat#: KC-0578; expressing EGFR and PD-L1) were cultured at 5.0 × 10⁻⁶ cells / year. 5 Cells were suspended in DMEM medium at a density of 10 cells / mL, and then 80 μL of the cell suspension was added to each well of a 96-well plate. The antibody to be tested was serially diluted (starting concentration 200 μg / mL, 3.16-fold serial dilutions) and added to each well at 10 μL, and incubated for 30 minutes. Then, 10 μL of human AB serum (ORi CELLS, Cat#: 20230419-Z0575) was added to each well, mixed, and incubated for 3 hours. Cells were transferred to ice, centrifuged at low temperature to remove the supernatant, resuspended in FACS buffer, and then incubated with 7-AAD for 10 minutes.

[0310] Flow cytometry was used to analyze 5000 cells per well. Data analysis used Flowjo software to calculate the 7-AAD+ proportion of the target cell population, and GraphPad Prism to plot antibody vs. cell death ratio curves and calculate IC50. Results are shown in Table 23 and... Figure 21 .

[0311] Table 23. Killing activity of EGFR×PDL1 bispecific antibody and control antibody against tumor cells (via CDC action)

[0312] Antibody IC50 (μg / mL) Cetuximab 2.04 H103G9x58E3V4-hIgG1-new 0.74 H22A12x58E3V4-hIgG1-new 0.63

[0313] 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, the amino acid sequence of which comprises: the amino acid sequence shown in SEQ ID NO. 29; the amino acid sequence shown in SEQ ID NO. 30; and the amino acid sequence shown in SEQ ID NO.

31.

2. The polypeptide according to claim 1, characterized in that, The polypeptide comprises 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.15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

15. Preferably, the polypeptide comprises the amino acid sequence shown in SEQ ID NO.38 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.22 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

22.

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

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

5. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising the polypeptide of claim 1 or 2; Preferably, the antibody or its antigen-binding fragment comprises the polypeptide of claim 1 or 2 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.

6. The antibody or its antigen-binding fragment according to claim 5, 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.26, HCDR2 containing the amino acid sequence shown in SEQ ID NO.27, HCDR3 containing the amino acid sequence shown in SEQ ID NO.28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31; (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO.32, HCDR2 containing the amino acid sequence shown in SEQ ID NO.33, HCDR3 containing the amino acid sequence shown in SEQ ID NO.28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 34, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 35, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31; or (4) HCDR1 containing the amino acid sequence shown in SEQ ID NO.34, HCDR2 containing the amino acid sequence shown in SEQ ID NO.37, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.

31.

7. The antibody or its antigen-binding fragment according to claim 5 or 6, characterized in that, The antibody 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 each comprise: (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; (2) 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. 12; (3) 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. 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 11; (4) 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. 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 15; (5) The amino acid sequence shown in SEQ ID NO. 16 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. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; or (6) The amino acid sequence shown in SEQ ID NO.17 or an amino acid sequence having at least 75% identity with 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.

15.

8. The antibody or antigen-binding fragment thereof according to any one of claims 5 to 7, 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: LCDR1 containing the amino acid sequence shown in SEQ ID NO.29; LCDR2 containing the amino acid sequence shown in SEQ ID NO.30; and LCDR3 containing the amino acid sequence shown in SEQ ID NO.

31. 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 or SEQ ID NO.15 or an amino acid sequence having at least 75% identity with the amino acid sequence.

9. The antibody or antigen-binding fragment thereof according to any one of claims 5 to 8, characterized in that, The antibody or its antigen-binding fragment comprises: (1) The first VH+VL domain combination of EGFR, wherein the first VH+VL domain combination comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 26, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 27, and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 29, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 containing SEQ ID NO. 31; and Combining the second VH+VL domain combination of PD-L1, the second VH+VL domain combination comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 34, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 37, 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. 29, LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 containing SEQ ID NO. 31; or (2) The first VH+VL domain combination of EGFR, wherein the first VH+VL domain combination comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 32; HCDR2 containing the amino acid sequence shown in SEQ ID NO. 33; and HCDR3 containing the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO. 29; LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30; and LCDR3 containing the amino acid sequence shown in SEQ ID NO. 31; and The second VH+VL domain combination of PD-L1 comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO. 34, HCDR2 containing the amino acid sequence shown in SEQ ID NO. 37, 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. 29; LCDR2 containing the amino acid sequence shown in SEQ ID NO. 30; and LCDR3 containing the amino acid sequence shown in SEQ ID NO.

31.

10. The antibody or antigen-binding fragment thereof according to any one of claims 5 to 9, characterized in that, The antibody or its antigen-binding fragment comprises: (1) A combination of the first VH+VL domains of EGFR, wherein the first VH+VL domain combination comprises: a heavy 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 a light chain variable region comprising 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 Combining the second VH+VL domain combination of PD-L1, the second VH+VL domain combination comprises: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO. 17 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. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; or (2) The first VH+VL domain combination of EGFR, wherein the first VH+VL domain combination comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 16 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. 15 or an amino acid sequence having at least 75% identity with said amino acid sequence; and The second VH+VL domain combination of PD-L1 includes: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO. 17 or an amino acid sequence having at least 75% identity with 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.

15.

11. The antibody or antigen-binding fragment thereof according to any one of claims 5 to 10, 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.

12. A composition comprising the polypeptide of claim 1 or 2, the nucleic acid molecule of claim 3, or the antibody or antigen-binding fragment thereof of any one of claims 5 to 11.

13. Use of the polypeptide of claim 1 or 2, the nucleic acid molecule of claim 3, the antibody or antigen-binding fragment thereof of any one of claims 5 to 11, or the composition of claim 12 in the preparation of a medicament.

14. 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: (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 26, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 27, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 28; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31; or (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO.32, HCDR2 containing the amino acid sequence shown in SEQ ID NO.33, HCDR3 containing the amino acid sequence shown in SEQ ID NO.28; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.

31.

15. An antibody or antigen-binding fragment thereof that binds to PD-L1, 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: (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 34, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 35, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 36; and LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, LCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31; or (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO.34, HCDR2 containing the amino acid sequence shown in SEQ ID NO.37, HCDR3 containing the amino acid sequence shown in SEQ ID NO.36; and LCDR1 containing the amino acid sequence shown in SEQ ID NO.29, LCDR2 containing the amino acid sequence shown in SEQ ID NO.30, and LCDR3 containing the amino acid sequence shown in SEQ ID NO.31.

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