Tumor dual-targeting t cell engager targeting EGFR and her2, and use thereof

Abstract

Provided are a tumor dual-targeting T cell engager (TDTCE) targeting EGFR and HER2, and the use thereof. The TDTCE targeting EGFR and HER2 has good specific T cell activating and proliferating activity and T cell anti-tumor activity at the cellular level, and has good efficacy and safety against solid tumors.

Description

[0001] The present application claims the priority of Chinese Patent Application No. 2024103228422 filed on March 20, 2024, and Chinese Patent Application No. 2025101429243 filed on February 8, 2025. The Chinese patent applications are incorporated herein by reference in their entireties. TECHNICAL FIELD

[0002] The present disclosure relates to the technical field of biopharmaceuticals, and in particular, to a tumor dual-targeting T cell engager targeting EGFR and HER2, and the use thereof. BACKGROUND

[0003] A T cell engager (TCE) is a class of biomacromolecular drugs that achieve specific killing of tumor cells based on the principle of T cell redirection. Existing marketed TCE drugs are mainly used in the field of hematological tumor treatment and have poor efficacy in solid tumors. The main reason is that in TCE drug development, the selection of tumor antigens for solid tumors is limited, and the selected antigens are mainly tumor-overexpressed antigens, such as HER2, EGFR, etc., while these antigens are also expressed at low levels in some important organs (such as the lung, heart, etc.), and potent therapeutic effects are likely to cause irreversible toxic side effects on these normal tissues. A tumor dual-targeting T cell engager (TDTCE) simultaneously binds two antigens on the surface of a tumor cell and the CD3 antigen on a T cell to mediate T cell anti-tumor activity, thereby reducing TCE-mediated cytotoxicity toward normal tissues expressing only a single antigen, and has great potential to improve the poor efficacy of conventional single-targeting TCEs against solid tumors.

[0004] For example, the patent application with publication No. CN 116239700 A discloses the preparation and use of a tumor dual-targeting trispecific T cell engager. The tumor dualtargeting trispecific T cell engager of the application has better specific T cell activation and proliferation activities and T cell anti-tumor activity at the cellular level, and can more effectively accumulate inside solid tumors, which is beneficial for the treatment of solid tumors.

[0005] HER2 and EGFR are "star" targets in tumors, co-expressed in most tumors, and are ideal targets for TDTCE. A trispecific T cell engager (THC) targeting HER2 and EGFR is under development (patent publication No. CN 118240093 A), and THC has potent and specific anti-tumor activity in vitro. However, as research progressed, it was found that although THC improved safety to some extent, there was still considerable room for improvement; particularly, the safety issues caused by targeting HER2 remained significant and were a key focus for improvement. Therefore, the TDTCE targeting HER2 and EGFR needs to be optimized and developed. SUMMARY

[0006] The present disclosure provides a tumor dual-targeting T cell engager (TDTCE) targeting EGFR and HER2, and the use thereof.

[0007] A first aspect of the present disclosure provides a tumor dual-targeting T cell engager, wherein the tumor dual-targeting T cell engager includes a first functional region, a second functional region, and a third functional region targeting EGFR, HER2, and CD3, respectively;

[0008] the tumor dual-targeting T cell engager has the following structure:

[0009] (1) the first functional region and the third functional region are scFv structures, the second functional region is a Fab structure, and a C-terminus of a light chain of the second functional region is connected to an N-terminus of the third functional region; or

[0010] (2) the first functional region is a Fab structure, the second functional region and the third functional region are scFv structures, and the C-terminus of the light chain of the first functional region is connected to the N-terminus of the third functional region.

[0011] In some embodiments, the scFv structure comprises a heavy-chain variable region (VH) and a light-chain variable region (VL) connected by a linker, and the scFv structure, from the N-terminus to the C-terminus, is: VL-linker-VH or VH-linker-VL.

[0012] In some embodiments, the Fab structure comprises a heavy-chain variable region (VH), a light-chain variable region (VL), a heavy chain constant region 1 (CH1), and a light chain constant region (CL), wherein the VH is connected to CH1, and the VL is connected to CL.

[0013] In some specific embodiments, the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 66-68.

[0014] In some embodiments, the CH1 and the CL are CH1 and CL of human IgG, respectively.

[0015] In some preferred embodiments, the CH1 has an amino acid sequence as set forth in SEQ ID NO: 132.

[0016] In some preferred embodiments, the CL has an amino acid sequence as set forth in SEQ ID NO: 65 or 133.

[0017] In some embodiments, the tumor dual-targeting T cell engager further includes an Fc, wherein the Fc is connected by a hinge region to the C-termini of the heavy-chain portions of the first functional region and the second functional region, respectively.

[0018] In some embodiments, the Fc has a knob-into-hole structure.

[0019] In some preferred embodiments, the Fc comprises L234A / L235A / P329G mutations.

[0020] In some preferred embodiments, the Fc further comprises H435R / Y436F mutations or H435R / Y436C mutations.

[0021] In the present disclosure, " / " indicates that the mutations before and after the " / " are present simultaneously.

[0022] In the present disclosure, the CDRs are defined according to the Kabat numbering system.

[0023] In some embodiments, the hinge region is a hinge region of human IgG.

[0024] In some preferred embodiments, the first cysteine (Cys) of the hinge region is substituted with serine (Ser).

[0025] In some embodiments, the first functional region targeting EGFR comprises a VH and a VL, wherein (1) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; or (2) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, respectively.

[0026] In some embodiments, the second functional region targeting HER2 comprises a VH and a VL, wherein (i) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or (ii) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 55, SEQ ID NO: 56, and HCDR3 SEQ ID NO: 57, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively.

[0027] In some embodiments, the third functional region targeting CD3 comprises a VH and a VL, wherein (a) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, respectively; or (b) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 43, SEQ ID NO: 117, and SEQ ID NO: 118, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 120, SEQ ID NO: 48, and SEQ ID NO: 121, respectively; or (c) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 124, SEQ ID NO: 117, and SEQ ID NO: 125, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively.

[0028] In some preferred embodiments, the first functional region targeting EGFR comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 37 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 37, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 41 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 41; or the first functional region targeting EGFR comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 111 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 111, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 115 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 115.

[0029] In some preferred embodiments, the first functional region targeting HER2 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 28 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 28, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 32 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 32; or the first functional region targeting HER2 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 58 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 58, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 62 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 62.

[0030] In some preferred embodiments, the first functional region targeting CD3 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 46 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 46, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 50 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 50; or the first functional region targeting CD3 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 119 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 119, and a VL , wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 122 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 122; or the first functional region targeting CD3 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 126 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 126, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 130 or is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 130.

[0031] In some preferred embodiments, when the first functional region is the scFv structure, the first functional region has an amino acid sequence as set forth in SEQ ID NO: 42 or 116 or has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 42 or 116.

[0032] In some preferred embodiments, when the second functional region is the scFv structure, the first functional region has an amino acid sequence as set forth in SEQ ID NO: 33 or 63 or has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 33 or 63.

[0033] In some preferred embodiments, when the third functional region is the scFv structure, the first functional region has an amino acid sequence as set forth in any one of SEQ ID NOs: 51, 123, and 131 or has an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NOs: 51, 123, and 131.

[0034] In some embodiments, the tumor dual-targeting T cell engager includes a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain;

[0035] the structure of the first polypeptide chain, from the N-terminus to the C-terminus, is: VL-CL-scFv1;

[0036] the structure of the second polypeptide chain, from the N-terminus to the C-terminus, is: VH-CH1-Fc1;

[0037] the structure of the third polypeptide chain, from the N-terminus to the C-terminus, is: scFv2-Fc2;

[0038] the VH and VL, scFv1, and scFv2 target different antigens, wherein the antigens are selected from EGFR, HER2, and CD3; Fc1 and Fc2 are identical or form a knob-into-hole structure.

[0039] In some preferred embodiments, the structure of the scFv1, from the N-terminus to the C-terminus, is: VL1-linker-VH1, and the structure of the scFv2, from the N-terminus to the C-terminus, is: VL2-linker-VH2.

[0040] In some embodiments, the VH and VL target EGFR or HER2, the scFv1 targets CD3, and the scFv2 targets HER2 or EGFR.

[0041] In some preferred embodiments, the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from any one of the following structures:

[0042] (1) the first polypeptide chain: EGFR VL-CL-CD3 scFv; the second polypeptide chain: EGFR VH-CH1-Fc1; the third polypeptide chain: HER2 scFv-Fc2;

[0043] (2) the first polypeptide chain: HER2 VL-CL-CD3 scFv; the second polypeptide chain: HER2 VH-CH1-Fc1; the third polypeptide chain: EGFR scFv-Fc2.

[0044] In some specific embodiments, the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 2; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 1; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 3; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 134; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 87; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84.

[0045] In some specific embodiments, the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 79; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 78; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 79; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 78; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 79; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 90; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84.

[0046] In some specific embodiments, the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 9; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 8; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 3; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 77; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 86.

[0047] In some specific embodiments, the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 80; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 80; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 80; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 87; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84.

[0048] In some specific embodiments, the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 82; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 82; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 82; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 87; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84.

[0049] A second aspect of the present disclosure provides a trispecific T cell engager targeting HER2 and EGFR, including a backbone comprising an IgG antibody constant region, wherein the backbone includes two heavy chains and one light chain, wherein one of the heavy chains includes heavy chain constant regions CH1, CH2, and CH3, the other heavy chain includes CH2 and CH3, the light chain includes CL, and the trispecific T cell engager targeting HER2 and EGFR is a heterotrimer, specifically:

[0050] the first subunit includes the light-chain variable region of an anti-HER2 antibody, the light chain in the backbone, as well as the heavy-chain variable region and light-chain variable region of an anti-CD3 antibody; the second subunit includes the heavy-chain variable region of the anti-HER2 antibody and one heavy chain in the backbone; and the third subunit includes the heavy-chain variable region and light-chain variable region of an anti-EGFR antibody as well as the other heavy chain in the backbone;

[0051] wherein the light-chain variable region of the anti-HER2 antibody in the first subunit and the heavy-chain variable region of the anti-HER2 antibody in the second subunit form an anti-HER2 antibody domain, the heavy-chain variable region and light-chain variable region of the anti-CD3 antibody in the first subunit form an anti-CD3 antibody domain, and the heavychain variable region and light-chain variable region of the anti-EGFR antibody in the third subunit form an anti-EGFR antibody domain.

[0052] In the present disclosure, the trispecific T cell engager is preferably a tumor dualtargeting T cell engager.

[0053] The IgG antibody is a human IgG antibody.

[0054] In the examples of the present disclosure, as an illustration, the anti-CD3 antibody is selected to be an anti-CD3s antibody. However, the present disclosure is not limited thereto.

[0055] Preferably, the two CH3 domains in the second subunit and the third subunit are respectively designed as a knob structure and a hole structure using the knob-into-hole technology. LALA-PG mutations are introduced into the CH2 domains of the second subunit and the third subunit. The mutations silence the antibody Fc fragment and abolish its ability to bind Fc receptors, thereby avoiding the toxic side effects of Fc-mediated nonspecific killing.

[0056] Specifically, when the heavy-chain variable region and light-chain variable region of the anti-CD3 antibody are connected to the first subunit, the amino acid sequence of the first subunit is set forth in SEQ ID NO: 2 or SEQ ID NO: 9, the amino acid sequence of the second subunit is set forth in SEQ ID NO: 1 or SEQ ID NO: 8, and the amino acid sequence of the third subunit is set forth in SEQ ID NO: 3.

[0057] A third aspect of the present disclosure provides a tumor dual-targeting T cell engager targeting EGFR and HER2, including a backbone comprising an IgG antibody constant region, wherein the backbone includes two heavy chains and one light chain, wherein one of the heavy chains includes heavy chain constant region CH1, a hinge region, CH2, and CH3, the other heavy chain includes a hinge region, CH2, and CH3, the light chain includes CL, and the tumor dual-targeting T cell engager targeting EGFR and HER2 is a trispecific antibody, specifically:

[0058] the first subunit includes the light-chain variable region of an anti-EGFR antibody, the light chain in the backbone, as well as the heavy-chain variable region and light-chain variable region of an anti-CD3 antibody; the second subunit includes the heavy-chain variable region of the anti-EGFR antibody and one heavy chain in the backbone; and the third subunit includes the heavy-chain variable region and light-chain variable region of an anti-HER2 antibody as well as the other heavy chain in the backbone;

[0059] wherein the light-chain variable region of the anti-EGFR antibody in the first subunit and the heavy-chain variable region of the anti-EGFR antibody in the second subunit form an anti-EGFR antibody domain, the light-chain variable region and heavy-chain variable region of the anti-CD3 antibody in the first subunit form an anti-CD3 antibody domain, and the lightchain variable region and heavy-chain variable region of the anti-HER2 antibody in the third subunit form an anti-HER2 antibody domain.

[0060] The IgG antibody is a human IgG antibody.

[0061] In the examples of the present disclosure, as an illustration, the anti-CD3 antibody is selected to be an anti-CD3c antibody. However, the present disclosure is not limited thereto.

[0062] Preferably, the amino acid sequence of the light-chain variable region of the anti-EGFR antibody in the first subunit consists of amino acids from position 1 to position 107 of SEQ ID NO: 3 or SEQ ID NO: 71, and the amino acid sequence of the heavy-chain variable region and light-chain variable region of the anti-CD3 antibody consists of amino acids from position 230 to position 483 of SEQ ID NO: 2, amino acids from position 232 to position 485 of SEQ ID NO: 6, or amino acids from position 230 to position 483 of SEQ ID NO: 9; the amino acid sequence of the heavy-chain variable region of the anti-EGFR antibody in the second subunit consists of amino acids from position 123 to position 241 of SEQ ID NO: 3; the amino acid sequence of the heavy-chain variable region and light-chain variable region of the anti-HER2 antibody in the third subunit consists of amino acids from position 1 to position 120 of SEQ ID NO: 72 or amino acids from position 1 to position 107 of SEQ ID NO: 77.

[0063] Preferably, in the present disclosure, as an illustration, the two CH3 domains in the second subunit and the third subunit are respectively designed as a hole structure and a knob structure using the knob-into-hole technology. LALA-PG mutations are introduced into the CH2 domains of the second subunit and the third subunit. The mutations silence the antibody Fc fragment and abolish its ability to bind Fc receptors, thereby avoiding the toxic side effects of Fc-mediated nonspecific killing. RF mutations or RC mutations are introduced into the CH3 domain of the second subunit to reduce the binding of this chain to protein A; furthermore, the first cysteine of the hinge region in the third subunit is mutated to serine.

[0064] Preferably, after the design of a knob structure and a hole structure, the introduction of LALA-PG mutations, and the introduction of RF mutations or RC mutations into the CH3 domain of the second subunit, the amino acid sequence of one heavy chain in the backbone in the second subunit consists of amino acids from position 220 to position 449 of SEQ ID NO: 70; the amino acid sequence of the other heavy chain in the backbone in the third subunit consists of amino acids from position 253 to position 483 of SEQ ID NO: 72 or amino acids from position 252 to position 482 of SEQ ID NO: 77.

[0065] The tumor dual-targeting T cell engager provided by the present disclosure, on the premise of maintaining the basic structure of the IgG antibody backbone, allows various reasonable mutations and modifications to the backbone. These mutations and modifications do not adversely affect the performance of the engager and instead help optimize its safety and efficacy. By introducing LALA-PG mutations to silence the Fc fragment, the toxic side effect of non-specific killing is avoided; by introducing RF mutations or RC mutations to reduce the binding to protein A, the purification efficiency is improved; and the first cysteine of the hinge region in the third subunit is mutated to serine to maintain the structural stability and activity of the engager. In the examples, we verified the effects of these mutations on the performance of the engager, and the results indicated that the expression levels and activities were not significantly different. Therefore, the scope of protection of the present disclosure not only encompasses engagers having a wild-type backbone but also includes backbones subjected to reasonable mutations and modifications, provided that these mutations and modifications maintain the basic structure and function of the engager. Meanwhile, the present disclosure further encompasses engagers having different combinations of antibody domains, wherein these domains are connected by the backbone to jointly perform the functions of targeting EGFR and HER2 as well as activating T cells.

[0066] Specifically, the amino acid sequence of the first subunit is set forth in SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 29, the amino acid sequence of the second subunit is set forth in SEQ ID NO: 8, SEQ ID NO: 25, or SEQ ID NO: 33, and the amino acid sequence of the third subunit is set forth in SEQ ID NO: 10, SEQ ID NO: 24, SEQ ID NO: 31, or SEQ ID NO: 32.

[0067] A fourth aspect of the present disclosure provides a polynucleotide, wherein the polynucleotide encodes the tumor dual-targeting T cell engager according to the first aspect.

[0068] In some embodiments, the polynucleotide comprises a first nucleic acid, a second nucleic acid, and a third nucleic acid; the first nucleic acid, the second nucleic acid, and the third nucleic acid encode the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain, respectively.

[0069] In some preferred embodiments, the first nucleic acid has a sequence as set forth in any one of SEQ ID NOs: 12, 19, 92, 99, 100, and 102; the second nucleic acid has a sequence as set forth in any one of SEQ ID NOs: 11, 18, 91, 98, 106, 107, and 135; the third nucleic acid has a sequence as set forth in any one of SEQ ID NOs: 13, 93, 97, 104, and 105.

[0070] A fifth aspect of the present disclosure provides a gene encoding the trispecific T cell engager according to the second aspect.

[0071] In some embodiments of the present disclosure, the nucleotide sequence of the first subunit is set forth in SEQ ID NO: 12 or SEQ ID NO: 19, the nucleotide sequence of the second subunit is set forth in SEQ ID NO: 11 or SEQ ID NO: 18, and the nucleotide sequence of the third subunit is set forth in SEQ ID NO: 13.

[0072] A sixth aspect of the present disclosure provides a gene encoding the tumor dualtargeting T cell engager according to the third aspect.

[0073] Preferably, the nucleotide sequence of the light-chain variable region of the anti-EGFR antibody in the first subunit is the sequence at bp 1-321 of SEQ ID NO: 92 or SEQ ID NO: 99, and the nucleotide sequence of the heavy-chain variable region and light-chain variable region of the anti-CD3 antibody is the sequence at bp 688-1449 of SEQ ID NO: 12, the sequence at bp 694-1455 of SEQ ID NO: 16, or the sequence at bp 688-1449 of SEQ ID NO: 99; the nucleotide sequence of the heavy-chain variable region of the anti-EGFR antibody in the second subunit is the sequence at bp 1-357 of SEQ ID NO: 91 or SEQ ID NO: 99; the nucleotide sequence of the heavy-chain variable region and light-chain variable region of the anti-HER2 antibody in the third subunit is the sequence at bp 1-726 of SEQ ID NO: 93 or the sequence at bp 1-726 of SEQ ID NO: 97.

[0074] In the present disclosure, as an illustration, the two CH3 domains in the second subunit and the third subunit are respectively designed as a hole structure and a knob structure using the knob-into-hole technology; when LALA-PG mutations are introduced into the CH2 domains of the second subunit and the third subunit, and RF mutations or RC mutations are introduced into the CH3 domain of the second subunit, the nucleotide sequence of one heavy chain in the backbone in the second subunit is the sequence at bp 361-1350 of SEQ ID NO: 11 or the sequence at bp 358-1347 of SEQ ID NO: 18, and the nucleotide sequence of the other heavy chain in the backbone in the third subunit is the sequence at bp 754-1446 of SEQ ID NO: 13 or the sequence at bp 763-1455 of SEQ ID NO: 14.

[0075] Specifically, in some embodiments of the present disclosure, the nucleotide sequence of the first subunit is set forth in SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20, the nucleotide sequence of the second subunit is set forth in SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 18, and the nucleotide sequence of the third subunit is set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 93, or SEQ ID NO: 97.

[0076] A seventh aspect of the present disclosure provides a recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide according to the fourth aspect.

[0077] An eighth aspect of the present disclosure provides a transformant, wherein the transformant expresses the tumor dual-targeting T cell engager according to the first aspect, or comprises the polynucleotide according to the fourth aspect, or comprises the recombinant expression vector according to the seventh aspect.

[0078] A ninth aspect of the present disclosure provides a method for preparing a tumor dualtargeting T cell engager, wherein the method comprises culturing the transformant according to the eighth aspect, and obtaining the tumor dual-targeting T cell engager from the culture.

[0079] A tenth aspect of the present disclosure provides a method for preparing the trispecific T cell engager, including the following steps: (1) constructing coding gene vectors for expressing the coding gene of the first subunit, the coding gene of the second subunit, and the coding gene of the third subunit; (2) transfecting the coding gene vectors for expressing the coding gene of the first subunit, the coding gene of the second subunit, and the coding gene of the third subunit of step (1) into mammalian cells, culturing the mammalian cells, and then performing protein purification to obtain the trispecific T cell engager. Preferably, the vector in step (1) is pLVX-Puro; the mammalian cells in step (2) are Expi293F cells.

[0080] An eleventh aspect of the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the tumor dual-targeting T cell engager according to the first aspect or the third aspect, the trispecific T cell engager according to the second aspect, the polynucleotide according to the fourth aspect, the gene according to the fifth aspect or the sixth aspect, the recombinant expression vector according to the seventh aspect, and / or the transformant according to the eighth aspect, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0081] A twelfth aspect of the present disclosure provides use of the tumor dual-targeting T cell engager according to the first aspect or the third aspect, the trispecific T cell engager according to the second aspect, the polynucleotide according to the fourth aspect, the gene according to the fifth aspect or the sixth aspect, the recombinant expression vector according to the seventh aspect, the transformant according to the eighth aspect, or the pharmaceutical composition according to the eleventh aspect in the preparation of an anti-tumor drug.

[0082] The twelfth aspect of the present disclosure further provides use of the tumor dualtargeting T cell engager according to the first aspect or the third aspect, the trispecific T cell engager according to the second aspect, the polynucleotide according to the fourth aspect, the gene according to the fifth aspect or the sixth aspect, the recombinant expression vector according to the seventh aspect, the transformant according to the eighth aspect, or the pharmaceutical composition according to the eleventh aspect in the preparation of a drug for treating a cancer.

[0083] A thirteenth aspect of the present disclosure provides an anti-tumor drug, wherein the active ingredient is the tumor dual-targeting T cell engager according to the first aspect or the third aspect, the trispecific T cell engager according to the second aspect, or the gene or the polynucleotide. The types of tumors include tumor cells that are HER2 and EGFR doublepositive, and the tumor cells include colon cancer cells, melanoma cells, non-small cell lung cancer cells, and the like.

[0084] A fourteenth aspect of the present disclosure provides the tumor dual-targeting T cell engager according to the first aspect or the third aspect, the trispecific T cell engager according to the second aspect, the polynucleotide according to the fourth aspect, the gene according to the fifth aspect or the sixth aspect, the recombinant expression vector according to the seventh aspect, the transformant according to the eighth aspect, the pharmaceutical composition according to the eleventh aspect, or the anti-tumor drug according to the thirteenth aspect, for use in treating a cancer.

[0085] In some embodiments, the cancer is selected from breast cancer, gastric cancer, nonsmall cell lung cancer, colon cancer, epidermoid carcinoma, and melanoma.

[0086] A fifteenth aspect of the present disclosure provides a method for treating cancer, wherein the method includes administering to a subject in need thereof an effective amount of the tumor dual-targeting T cell engager according to the first aspect or the third aspect, the trispecific T cell engager according to the second aspect, the polynucleotide according to the fourth aspect, the gene according to the fifth aspect or the sixth aspect, the recombinant expression vector according to the seventh aspect, the transformant according to the eighth aspect, the pharmaceutical composition according to the eleventh aspect, or the anti-tumor drug according to the thirteenth aspect.

[0087] In some embodiments, the cancer is selected from breast cancer, gastric cancer, nonsmall cell lung cancer, colon cancer, epidermoid carcinoma, and melanoma.

[0088] Beneficial effects of the present disclosure:

[0089] The TDTCE of the present disclosure targeting EGFR and HER2 has good specific T cell activating and proliferating activity and T cell anti-tumor activity at the cellular level, and has good efficacy and safety against solid tumors. Therefore, the tumor dual-targeting T cell engager of the present disclosure or the gene encoding the same can be used for preparing an anti-tumor drug, and has the advantages of simple preparation and expression by mammalian cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] FIG. 1 shows a schematic diagram of the molecular format of the trispecific T cell engager targeting HER2 and EGFR (aHER2-aCD3 / aEGFR).

[0091] FIG. 2 shows an SDS-PAGE analysis of aHER2-aCD3 / aEGFR-1 (abbreviated as THC) and control molecules thereof.

[0092] FIG. 3 shows an affinity analysis of THC and control molecules thereof for CHO-EGFR (A), 4T1-HER2 (B), and Jurkat (C) cells.

[0093] FIG. 4 shows THC-mediated PBMC killing of tumor cells; (A) shows a comparison of the activity of THC and control molecules thereof in mediating PBMC killing of A431 cells; (B) shows a comparison of the activity of THC and control molecules thereof in mediating PBMC killing of A549 cells; (C) shows a comparison of the activity of THC and control molecules thereof in mediating PBMC killing of COLO205 cells; (D) shows a comparison of the activity of THC and control molecules thereof in mediating PBMC killing of AGS cells; the normalized OD490 represents the ratio of the OD490 of each group to the OD490 of the untreated control group.

[0094] FIG. 5 shows THC-stimulated specific T cell activation; (A) shows the proportion of T cells exhibiting early activation; (B) shows the proportion of T cells exhibiting late activation; significant differences between THC and other control groups at the same concentration: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

[0095] FIG. 6 shows an assessment of the anti-tumor activity of THC in an ATC-reconstituted mouse xenograft model; (A) shows the mean tumor volume of mice; (B) shows photographs of mouse tumors, with tails shown instead for the tumor-free group; (C) shows the body weight measurement of mice.

[0096] FIG. 7 shows an assessment of the anti-tumor activity of THC in a PBMC-reconstituted mouse xenograft model.

[0097] FIG. 8 shows an assessment of the in vitro specific anti-tumor activity of THC and aHER2-aCD3 / aEGFR-2 (abbreviated as PHC).

[0098] FIG. 9 shows a schematic diagram of the molecular format of the TDTCE targeting EGFR and HER2 (aEGFR-aCD3 / aHER2).

[0099] FIG. 10 shows an SDS-PAGE analysis of aEGFR-aCD3 / aHER2-1 (abbreviated as CHT) and control molecules thereof.

[0100] FIG. 11 shows an affinity analysis of CHT and control molecules thereof for CHO-EGFR cells (A), CHO-HER2 cells (B), Jurkat cells (C), human PBMCs (D), and cynomolgus monkey PBMCs (E).

[0101] FIG. 12 shows the results of CHT-stimulated specific T cell activation.

[0102] FIG. 13 shows the results of CHT-stimulated specific T cell proliferation.

[0103] FIG. 14 shows CHT-mediated human PBMC killing of tumor cells; (A) shows a comparison of the activity of CHT, CHT-2 and control molecules thereof, and THC and control molecules thereof in mediating PBMC killing of COLO205 cells; (B) shows a comparison of the activity of CHT and control molecules thereof in mediating PBMC killing of A375 cells; (C) shows a comparison of the activity of CHT and control molecules thereof in mediating PBMC killing of H460 cells; the normalized OD490 represents the ratio of the OD490 of each group to the OD490 of the untreated control group.

[0104] FIG. 15 shows an assessment of the anti-tumor activity and toxicity of CHT in an ATC-reconstituted mouse xenograft model.

[0105] FIG. 16 shows a safety assessment of the TDTCE targeting EGFR and HER2 in a HER2-transgenic mouse xenograft model; (A) shows the mean body weight change of mice; (B) shows the results of H&E histochemical staining of mouse organs.

[0106] FIG. 17 shows an assessment of the in vitro specific anti-tumor activity of aEGFR-aCD3 / aHER2-3 (abbreviated as CHP).

[0107] FIG. 18 shows an assessment of the in vitro specific anti-tumor activity of aEGFR-aCD3 / aHER2-4 (abbreviated as PHT).

[0108] FIG. 19 shows an assessment of the in vitro specific anti-tumor activity of aEGFR-aCD3 / aHER2-5 (abbreviated as CST) and aEGFR-aCD3 / aHER2-6 (abbreviated as CIT).

[0109] FIG. 20 shows an assessment of the in vitro anti-tumor activity of aEGFR- aCD3 / aHER2 before and after hinge region modification.

[0110] FIG. 21 shows an assessment of the in vitro anti-tumor activity of aEGFR- aCD3 / aHER2 with RF and RC modifications of the Fc fragment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0111] Definitions

[0112] As used herein, the terms "complementarity determining region," "CDR region," or "CDR" refer to a region of an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and / or contains an antigen-contact residue ("antigen contact point"). CDRs are mainly responsible for binding to antigens, and include, sequentially numbered starting from the N-terminus, CDR1, CDR2 and CDR3. In a given heavy-chain variable region amino acid sequence, the precise amino acid sequence boundary of each CDR may be determined by using any one of many well-known antibody CDR assignment systems or a combination thereof. It is well known to those skilled in the art that antibody CDRs may be defined by various methods, for example, Chothia, which is based on the three-dimensional structure of an antibody and the topology of the CDR loops; Kabat, AbM, Contact, and IMGT, which are based on antibody sequence variability; as well as the North CDR definition, which is based on affinity propagation clustering utilizing a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or a region thereof (for example, a variable region) should be understood to encompass complementarity determining regions as defined by any of the foregoing known schemes described herein.

[0113] Antibodies having different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the methods of Kabat, Chothia, IMGT, AbM, and Contact, a minimum overlapping region can be determined, thereby providing a "minimal binding unit" for antigen binding. The minimal binding unit may be a subpart of a CDR. As will be apparent to those skilled in the art, the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Therefore, the present disclosure also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by amino acid residues.

[0114] As used herein, the "percent (%) sequence identity" and "sequence identity" of an amino acid sequence have a definition recognized in the art and refer to the percentage of identity between two polypeptide sequences as determined by sequence alignment (for example, by human inspection or by well-known algorithms). The "percent (%) sequence identity" and "sequence identity" can be determined using methods known to those skilled in the art, for example, using publicly available computer software, e.g., BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0115] The following examples are described to assist in the understanding of the present disclosure. The examples are not intended and should not be construed to limit the scope of protection of the present disclosure in any way. Example 1: Preparation of a Trispecific TCE Targeting HER2 and EGFR

[0116] The molecular format of the trispecific TCE targeting HER2 and EGFR (called aHER2-aCD3 / aEGFR) is shown in FIG. 1. The following sequences were selected to express aHER2-aCD3 / aEGFR-1 (abbreviated as THC): Trastuzumab (abbreviated as Tra) was selected as the anti-HER2 antibody; its light-chain variable-region nucleotide sequence was obtained from NCBI, accession no. OP892523.1, and its heavy-chain variable-region nucleotide sequence was obtained from NCBI, accession no. OP892522.1; Cetuximab (abbreviated as CTX) was selected as the anti-EGFR antibody; its light-chain variable-region nucleotide sequence was obtained from NCBI, accession no. KX138561, and its heavy-chain variableregion nucleotide sequence was obtained from NCBI, accession no. KX138562.1; HXR32, disclosed in the following patent, was selected as the anti-CD3 antibody: WO 2012162067 A2. The control molecule sequences were replaced with the antibody D10-7 targeting Kras G12V / A2 (abbreviated as G12V, the variable region sequences were from NCBI, PMID: 26216968).

[0117] THC consists of three subunits: Tra-H-hole-LALAPG-Flag (the gene sequence of Tra-H-hole-LALAPG is as set forth in SEQ ID NO: 11, and the gene sequence of Flag is as set forth in SEQ ID NO: 21), Tra-L-HXR32 (the gene sequence is as set forth in SEQ ID NO: 12), and CTX-scFv-knob-His6 (the gene sequence of CTX-scFv-knob is as set forth in SEQ ID NO: 13, and the gene sequence of His6 is as set forth in SEQ ID NO: 22); the control molecule THG consists of three subunits: Tra-H-hole-LALAPG-Flag, Tra-L-HXR32, and G12V-scFv-knob-His6 (the gene sequence of G12V-scFv-knob is as set forth in SEQ ID NO: 14); the control molecule GHC consists of three subunits: G12V-H-hole-LALAPG-Flag (the gene sequence of G12V-H-hole-LALAPG is as set forth in SEQ ID NO: 15), G12V-L-HXR32 (the gene sequence is as set forth in SEQ ID NO: 16), and CTX-scFv-knob-His6; the control molecule TGC consists of three subunits: Tra-H-hole-LALAPG-Flag, Tra-L-G12V (the gene sequence is as set forth in SEQ ID NO: 17), and CTX-scFv-knob-His6.

[0118] His6 is a tag consisting of 6 histidine for protein purification (SEQ ID NO: 24), the Flag tag is a tag consisting of 8 amino acids DYKDDDDK (SEQ ID NO: 23) for protein purification, CL (human k light chain constant region sequence, GenBank No.: M63438), CH1, CH2, and CH3 are all structures of IgG1 (GenBank No.: P01857), and the two CH3 domains were respectively designed as a knob structure and a hole structure using the knob-into-hole technology, and simultaneously, the LALA-PG mutations were introduced into CH2 to silence the Fc.

[0119] Gene constructs encoding Tra-H-hole-LALAPG-Flag, Tra-L-HXR32, CTX-scFv-knob-His6, G12V-scFv-knob-His6, G12V-H-hole-LALAPG-Flag, G12V-L-HXR32, and Tra-L- G12V in pLVX-Puro plasmids (Beijing Tsingke Biotech Co., Ltd.) were synthesized and transiently transfected into Expi293F cells (purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. SCSP-5207). The expressed products were purified by column chromatography using a HisCap 6FF affinity column (purchased from SmartLifesciences Biotechnology Co., Ltd., Cat. No. SA005C55) and Anti-DYKDDDDK Affinity Beads (purchased from Smart-Lifesciences Biotechnology Co., Ltd., Cat. No. SA042005) to obtain the corresponding proteins, which were then analyzed by SDS-PAGE. As shown in FIG. 2, each subunit of THC and its control molecules appeared at the position corresponding to its theoretical size (the theoretical molecular weight of each subunit is shown in Table 1) and exhibited relatively high purity.

[0120] Table 1 Theoretical molecular weight of each subunit of the trispecific TCE targeting HER2 and EGFR Trispecific TCE Subunit Molecular weight (kDa) THC Tra-H-hole-LALAPG-Flag about 50 KDa Tra-L-HXR32 about 52 KDa CTX-scFv-knob-His6 about 53 KDa THG Tra-H-hole-LALAPG-Flag about 50 KDa Tra-L-HXR32 about 52 KDa G12V-scFv-knob-His6 about 53 KDa GHC G12V-H-hole-LALAPG-Flag about 50 KDa G12V-L-HXR32 about 52 KDa CTX-scFv-knob-His6 about 53 KDa TGC Tra-H-hole-LALAPG-Flag about 50 KDa Tra-L-G12V about 52 KDa CTX-scFv-knob-His6 about 53 KDa Example 2: Affinity Analysis of THC for HER2, EGFR, and CD3 Antigens

[0121] CHO-EGFR cells (EGFR-stably-transfected hamster ovary cells CHO-K1 (CHO-K1 purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. GNHa 7)), 4T1-HER2 cells (HER2-stably-transfected mouse breast cancer cells 4T1 (4T1 purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. SCSP-5056)), and human T lymphocyte leukemia cells Jurkat (CD3+ cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. SCSP-513) in the logarithmic growth phase were collected by centrifugation at 500 x g for 5 min, and then were respectively incubated with 100 liL of different concentrations of THC and control molecules thereof at 4°C for 30 min (with PBS incubation as a control). After washing twice with PBS, the cells were incubated with 100 lL of FITC-labeled goat anti-human IgG(H+L) (purchased from Beyotime Biotechnology Co., Ltd., Cat. No. A0556) at 4°C. After two washes with PBS, the cells were resuspended in 400 lL of PBS, and detection was performed using a BD FACSDiva flow cytometer.

[0122] As shown in FIG. 3, THC, TGC, and GHC exhibited similar affinities for CHO-EGFR cells in a concentration-dependent manner, whereas the EGFR-targeting antibody in THG was replaced with the G12V antibody, and THG therefore exhibited essentially no affinity for CHO-EGFR cells (FIG. 3A); THC, TGC, and THG exhibited similar affinities for 4T1-HER2 cells in a concentration-dependent manner, whereas the HER2-targeting antibody in GHC was replaced with the G12V antibody, and GHC therefore exhibited no affinity for 4T1-HER2 cells (FIG. 3B); THC, GHC, and THG exhibited similar affinities for Jurkat cells in a concentrationdependent manner, whereas the CD3-targeting antibody in TGC was replaced with the G12V antibody, and TGC therefore exhibited no affinity for Jurkat cells (FIG. 3C). Example 3: Assessment of the In Vitro Specific Anti-Tumor Activity of THC

[0123] HER2 / EGFR double-positive tumor cells (A431 epidermoid carcinoma cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. TCHu188; A549 non-small cell lung cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. SCSP-503; COLO 205 colon cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. TCHu102; and AGS gastric cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. TCHu232) were separately seeded with human peripheral blood mononuclear cells (PBMCs; purchased from Shanghai Sailybio Co., Ltd., Cat. No. XFWBC6072) at a ratio of 1:4 in 96-well cell culture plates. Serial dilutions of THC and its control molecules were added (n = 3), mixed, and incubated at 37OC in a 5% CO2 incubator. After 48 h, the culture supernatant was collected and analyzed using a lactate dehydrogenase (LDH) detection kit (purchased from Beyotime Biotechnology Co., Ltd., Cat. No. C0017), and the EC50 value was calculated utilizing GraphPad software.

[0124] As shown in FIG. 4, THC exhibited potent PBMC-mediated cytotoxicity against the HER2 / EGFR double-positive A431, A549, and COLO205 cells, as well as AGS cells (as measured by LDH release upon cell lysis), in a concentration-dependent manner. Its cytotoxic activity was much greater than that of THG, GHC, and TGC, as well as the combination of THG and GHC (i.e., the combination of single-target TCEs), indicating that THC had specific in vitro cytotoxic activity with a "1+1>2" effect. Example 4: Detection of specific T cell activation by THC

[0125] AGS cells were seeded with PBMCs at a ratio of 1:4 in 48-well cell culture plates. Serial dilutions of THC and its control molecules were added (n = 3), mixed, and incubated at 37°C in a 5% CO2 incubator. After 48 h of culture, the cells were collected by centrifugation at 600 g for 5 min, washed once with 1x PBS, and resuspended in 1x PBS. The cells were then incubated at 4°C in the dark for 30 min with 2 pL of Pacific Blue-labeled anti-human CD3 antibody (purchased from BioLegend, Cat. No. 300330), 2 pL of APC-labeled anti-human CD25 antibody (purchased from BioLegend, Cat. No. 302610), and 1 pL of FITC-labeled antihuman CD69 antibody (purchased from BioLegend, Cat. No. 310904). After washing twice with 1x PBS, the cells were resuspended in 200 pL of 1x PBS, and the proportions of CD69positive and CD25-positive T cells (CD3-positive) were detected using a BD FACSDiva flow cytometer.

[0126] As shown in FIG. 5, CD69 and CD25 are major markers of early and late T-cell activation, respectively. Co-incubation of THC with AGS cells could specifically and significantly activate T cells, and the proportion of activated T cells was much higher than that obtained with the various control molecules as well as the combination of single-target TCEs. Even at the extremely low concentration of 10 pM, co-incubation of THC with AGS cells could still significantly activate T cells. Example 5: Assessment of the Anti-Tumor Activity of THC in a Mouse Xenograft Model Reconstituted with Activated Human T Cells (ATCs)

[0127] PBMCs were activated with 1 pg / mL anti-human CD3 antibody (purchased from BioLegend, Cat. No. 317326), 1 pg / mL anti-human CD28 antibody (purchased from BioLegend, Cat. No. 302934), and 300 IU IL-2 (purchased from Acnovia, Cat. No. AC52350), and the activated PBMC cells (called ATC) were obtained. 1.5 x 107 ATCs were injected via the tail vein into 6-week-old M-NSG female mice (purchased from Shanghai Model Organisms Center, Inc.), and simultaneously each mouse was subcutaneously administered IL-2 at 1000 IU / 100 pL. On the next day, 5 x 106 COLO205 cells were subcutaneously inoculated on the right axillary region of M-NSG mice, slightly toward the dorsal side, and the day of tumor inoculation was designated as Day 0. The mice were assigned to a PBS group, a THC 500 pg / kg group, a THC 200 pg / kg group, and a THC 80 pg / kg group, with 5 mice per group. The corresponding treatments were administered respectively by tail-vein injection on Days 2, 5, and 8, and each mouse simultaneously received 1,000 IU IL-2 in 100 pL by subcutaneous injection. Tumor growth was monitored during the study, tumor volume was calculated as length x width x width / 2, and mouse body weight was measured. When the mean tumor volume of the mice in the PBS group approached 1000 mm3, the mice were euthanized, and the tumors were dissected from the subcutaneous tissue and photographed.

[0128] As shown in FIG. 6, THC exhibited potent tumor-killing activity in the ATC-reconstituted tumor-bearing mouse model. Tumor inhibition was pronounced at all dose levels, and even the low dose (80 pg / kg) produced an extremely significant tumor-eradicating effect, with a tumor inhibition rate of 100%. Body weights in all treatment groups fluctuated only within a narrow range, indicating that THC had low toxicity and a favorable safety profile. Example 6: Assessment of the Anti-Tumor Activity of THC in a Mouse Xenograft Model Reconstituted with PBMCs

[0129] 107 PBMCs were injected via the tail vein into 6-week-old M-NSG female mice. 17 days later, 5 x 106 COLO205 cells were subcutaneously inoculated on the right axillary region of M-NSG mice, slightly toward the dorsal side, and the day of tumor inoculation was designated as Day 0. Mice in which human CD3-positive T cells accounted for > 5% of total T cells in the serum were assigned to a PBS group, a THC 400 pg / kg group, and a THC 80 pg / kg group, with 6 mice per group. The corresponding treatments were administered respectively by tail-vein injection on Days 4 and 7. Tumor growth was monitored during the study, tumor volume was calculated as length x width x width / 2, and mouse body weight was measured.

[0130] As shown in FIG. 7, a single dose of THC inhibited tumor growth, and the tumors in the THC treatment groups gradually disappeared after the second dose. THC at 80 ugkg was sufficient to eliminate the tumors, indicating that THC exhibited potent tumor-killing activity in the PBMC-reconstituted tumor-bearing mouse model. Example 7: Assessment of the In Vitro Specific Anti-Tumor Activity of THC and PHC

[0131] The anti-HER2 antibody was replaced with pertuzumab (abbreviated as Per; sequence obtained from NCBI, PDB: 1S78_D). Per-H-hole-LALAPG-Flag (the Per-H-hole-LALAPG gene sequence is as set forth in SEQ ID NO: 18), Per-L-HXR32 (the gene sequence is as set forth in SEQ ID NO: 19), and CTX-scFv-knob (the gene sequence is as set forth in SEQ ID NO: 13) were used to construct and express aHER2-aCD3 / aEGFR-2 (abbreviated as PHC) and its control molecules PHG, PGC, and GHC by the method described in Example 1. AGS cells and PBMCs were seeded at a ratio of 1:4 in 96-well cell culture plates. Serial dilutions of THC, PHC, and the PHC control molecules were added (n = 3), mixed, and incubated at 37OC in a 5% CO2 incubator. After 48 h, the culture supernatant was collected and analyzed using an LDH detection kit, and the EC50 value was calculated utilizing GraphPad software.

[0132] As shown in FIG. 8, PHC mediated PBMC killing of AGS cells to an extent similar to THC, and its killing activity was much greater than that of the control molecules PHG, PGC, and GHC, indicating that potent and specific anti-tumor activity similar to that of the parental molecule could likewise be obtained by replacing the antigen-targeting sequence of this class of trispecific T cell engagers targeting HER2 and EGFR. Example 8: Preparation of a Trispecific TCE Targeting EGFR and HER2 (TDTCE)

[0133] The molecular format of the TDTCE targeting EGFR and HER2 (called aEGFR-aCD3 / aHER2) is shown in FIG. 9. The following sequences were selected to express aEGFR-aCD3 / aHER2-1 (abbreviated as CHT): Cetuximab (abbreviated as CTX; its light-chain variable-region nucleotide sequence was obtained from NCBI, accession no. KX138561, and its heavy-chain variable-region nucleotide sequence was obtained from NCBI, accession no.KX138562.1) was selected as the anti-EGFR antibody; Trastuzumab (abbreviated as Tra; its light-chain variable-region nucleotide sequence was obtained from NCBI, accession no. OP892523.1, and its heavy-chain variable-region nucleotide sequence was obtained from NCBI, accession no.OP892522.1) was selected as the anti-HER2 antibody; HXR32, disclosed in the patent WO 2012162067 A2, was selected as the anti-CD3 antibody. The control molecule sequences were replaced with the antibody D10-7 targeting Kras G12V / A2 (abbreviated as G12V, the variable region sequences were from NCBI, PMID: 26216968).

[0134] CHT consists of three subunits: CTX-H-hole-LALAPG-RF (the gene sequence is as set forth in SEQ ID NO: 91), CTX-L-HXR32 (the gene sequence is as set forth in SEQ ID NO: 92), and Tra-scFv-knob (the gene sequence is as set forth in SEQ ID NO: 93); the control molecule CHG consists of three subunits: CTX-H-hole-LALAPG-RF, CTX-L-HXR32, and G12V-scFv-knob (the gene sequence is the same as that in the patent with publication No. CN 118240093 A); the control molecule GHT consists of three subunits: G12V-H-hole-LALAPG (the gene sequence is the same as that in the patent with publication No. CN 118240093 A), G12V-L-HXR32 (the gene sequence is the same as that in the patent with publication No. CN118240093A), and Tra-scFv-knob; the control molecule CGT consists of three subunits: CTX-H-hole-LALAPG-RF, CTX-L-G12V (the gene sequence is as set forth in SEQ ID NO: 94), and Tra-scFv-knob.

[0135] CL (the human k light-chain constant-region sequence; GenBank No. M63438), CH1, CH2, and CH3 are all IgG1 structures (GenBank No. P01857). LALA-PG mutations were introduced into CH2 to silence Fc, the two CH3 domains were engineered as knob and hole structures, respectively, using knob-into-hole technology, and RF mutations were introduced into CH3 of the hole-mutant chain to reduce binding of the chain to Protein A.

[0136] Gene constructs encoding CTX-H-hole-LALAPG-RF, CTX-L-HXR32, Tra-scFv-knob, G12V-scFv-knob, G12V-H-hole-LALAPG, G12V-L-HXR32, and CTX-L-G12V in pLVX-Puro plasmids (Beijing Tsingke Biotech Co., Ltd.) were synthesized and transiently transfected into CHO-S cells (purchased from Thermo Fisher Scientific, Cat. No. R80007). The expressed products were purified using a Protein A affinity column, AbCap A 4FF (purchased from Smart-Lifesciences Biotechnology Co., Ltd., Cat. No. SA015C15), and SEC to obtain the corresponding proteins, which were then analyzed by SDS-PAGE. As shown in FIG. 10, each subunit of CHT and its control molecules appeared at the position corresponding to its theoretical size (the theoretical molecular weight of each subunit is shown in Table 2) and exhibited relatively high purity.

[0137] Table 2 Theoretical molecular weight of each subunit of the TDTCE targeting EGFR and HER2 TDTCE Subunit Molecular weight (kDa) CHT CTX-H-hole-LALAPG-RF about 50 KDa CTX-L-HXR32 about 52 KDa Tra-scFv-knob about 53 KDa CHG CTX-H-hole-LALAPG-RF about 50 KDa CTX-L-HXR32 about 52 KDa G12V-scFv-knob about 53 KDa GHT G12V-H-hole-LALAPG about 50 KDa G12V-L-HXR32 about 52 KDa Tra-scFv-knob about 53 KDa CGT CTX-H-hole-LALAPG-RF about 50 KDa CTX-L-G12V about 52 KDa Tra-scFv-knob about 53 KDa Example 9: Affinity Analysis of CHT for EGFR, HER2, and CD3 Antigens

[0138] CHO-EGFR cells (EGFR-stably-transfected hamster ovary cells CHO-K1 (CHO-K1 purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. GNHa 7)), CHO-HER2 cells (HER2-stably-transfected CHO cells), human T lymphocyte leukemia cells Jurkat (CD3+ cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. SCSP-513), human PBMCs (purchased from Shanghai Sailybio Co., Ltd., Cat. No. XFWBC6044), and cynomolgus monkey PBMCs (purchased from Shanghai Sailybio Co., Ltd., Cat. No. CMP230402-186783C) in the logarithmic growth phase were collected by centrifugation at 500 x g for 5 min, and then were respectively incubated with 100 pL of different concentrations of CHT and control molecules thereof at 4OC for 30 min (with PBS incubation as a control). After washing twice with PBS, the cells were incubated with 100 pL of FITC-labeled goat anti-human IgG(H+L) (purchased from Beyotime Biotechnology Co., Ltd., Cat. No. A0556) at 4OC.  After two washes with PBS, the cells were resuspended in 400 pL of PBS, and detection was performed using a BD FACSDiva flow cytometer.

[0139] As shown in FIG. 11, CHT, CHG, and CGT exhibited affinity for CHO-EGFR cells in a concentration-dependent manner, whereas the EGFR-targeting antibody in GHT was replaced with the G12V antibody, and GHT therefore exhibited essentially no affinity for CHO-EGFR cells (FIG. 11A); CHT, CGT, and GHT exhibited similar affinities for CHO-HER2 cells in a concentration-dependent manner, whereas the HER2-targeting antibody in CHG was replaced with the G12V antibody, and CHG therefore exhibited essentially no affinity for CHO-HER2 cells (FIG. 11B); CHT, GHT, and CHG exhibited similar affinities for Jurkat cells in a concentration-dependent manner, whereas the CD3-targeting antibody in CGT was replaced with the G12V antibody, and CGT therefore exhibited no affinity for Jurkat cells (FIG. 11C). In addition, CHT, GHT, and CHG not only specifically bound human PBMCs (FIG. 11D) but also exhibited specific affinity for cynomolgus monkey PBMCs (FIG. 11E). Example 10: Detection of specific T cell activation by CHT

[0140] COLO205 cells (colon cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. TCHu102) and PBMCs were seeded at a ratio of 1:4 in 48-well cell culture plates. Serial dilutions of CHT and its control molecules were added (n = 3), mixed, and incubated at 37OC in a 5% CO2 incubator. After 48 h of culture, the cells were collected by centrifugation at 600 x g for 5 min, washed once with 1x PBS, and resuspended in 1x PBS. The cells were then incubated at 4OC in the dark for 30 min with 2 pL of Pacific Blue-labeled anti-human CD3 antibody (purchased from BioLegend, Cat. No. 300330), 2 pL of APC-labeled anti-human CD25 antibody (purchased from BioLegend, Cat. No. 302610), and 1 pL of FITC-labeled anti-human CD69 antibody (purchased from BioLegend, Cat. No. 310904). After washing twice with 1x PBS, the cells were resuspended in 200 pL of 1x PBS, and the proportions of CD69-positive and CD25-positive T cells (CD3-positive) were detected using a BD FACSDiva flow cytometer.

[0141] As shown in FIG. 12, CD69 and CD25 are major markers of early and late T-cell activation, respectively. Co-incubation of CHT with COLO205 cells could specifically and significantly activate T cells, and the proportion of activated T cells was much higher than that obtained with the various control molecules as well as the combination of single-target TCEs. Even at the extremely low concentration of 10 pM, co-incubation of CHT with COLO205 cells could still significantly activate T cells. Example 11: Detection of specific T cell proliferation by CHT

[0142] PBMCs were collected by centrifugation at 600 x g for 10 min, washed twice with 1x PBS prewarmed to room temperature, and resuspended in 1x PBS. CFSE (purchased from Thermo Fisher Scientific, Cat. No. 65-0850-84) was added to a final concentration of 2.5 pM, and the cells were mixed and incubated at 37OC in the dark for 15 min. COLO205 cells and CFSE-treated PBMCs were collected by centrifugation at 600 x g for 10 min and washed once with PBS. The COLO205 cells were then seeded with CFSE-treated PBMCs at a ratio of 1:4 in 48-well cell culture plates. Serial dilutions of CHT and its control molecules were added (n = 3), mixed, and incubated at 37OC in a 5% CO2 incubator. After 96 h of culture, the cells were collected by centrifugation at 600 x g for 5 min, washed twice with 1x PBS, and resuspended in 100 pL of 1x PBS. The cells were incubated at room temperature in the dark for 15 min with 2 pL of Pacific Blue-labeled anti-human CD3 antibody, 2 pL of APC-labeled anti-human CD8 antibody (purchased from Dakewe Biotech Co., Ltd., Cat. No. 301014), and 5 pL of PI stock solution (1 mg / mL; purchased from Beyotime Biotechnology Co., Ltd., Cat. No. ST511). The cells were then collected by centrifugation at 600 x g for 5 min, resuspended in 400 pL of 1x PBS, and T-cell proliferation was detected using a BD FACSDiva flow cytometer.

[0143] After T cells are activated, they can divide and proliferate. CFSE can bind nonspecifically to intracellular proteins and generate green fluorescence. During cell division, CFSE is equally partitioned between the two daughter cells, and the green fluorescence decreases stepwise with each successive division. As shown in FIG. 13, compared with the blank control group, co-incubation of CHT with COLO205 cells stimulated T-cell proliferation, and the proportion of proliferating cells was proportional to the treatment concentration; Compared with CHT, the control molecules as well as the combination of single-target TCEs stimulated T-cell proliferation upon co-incubation with COLO205 cells only at the high concentration of 1,000 pM, and the fold increase in proliferation was lower than that obtained with CHT, indicating that co-incubation of CHT with COLO205 cells strongly and specifically stimulated T-cell proliferation. In addition, CHT exerted a stronger proliferative effect on CD8-positive T cells. Example 12: Assessment of the In Vitro Specific Anti-Tumor Activity of CHT

[0144] HER2 / EGFR double-positive tumor cells (COLO 205 cells; A375 melanoma cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. TCHu155; and H460 non-small cell lung cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. TCHu205) were separately seeded with PBMCs at a ratio of 1:4 in 96-well cell culture plates. Serial dilutions of CHT; CHT-2, whose Fc lacks the knob-into-hole and LALA-PG mutations (GenBank No. P01857); the CHT control molecules GHT and CHG; THC disclosed in patent CN 118240093 A; or the THC control molecules THG, TGC, and GHC disclosed in patent CN 118240093 A were added (n = 3), mixed, and incubated at 37°C in a 5% CO2 incubator. After 48 h, the culture supernatant was collected and analyzed using a lactate dehydrogenase (LDH) detection kit (purchased from Beyotime Biotechnology Co., Ltd., Cat. No. C0017), and the EC50 value was calculated utilizing GraphPad software.

[0145] As shown in FIG. 14A, CHT and CHT-2 exhibited potent PBMC-mediated cytotoxicity against COLO205 cells (as measured by LDH release upon cell lysis) in a concentration-dependent manner, and their cytotoxic activities were comparable, indicating that the knob-into-hole mutations and Fc silencing had essentially no effect on PBMC-mediated cytotoxicity; the killing activity of CHT was far higher than that of CHG, GHT, and CGT, as well as the combination of CHG and GHT (i.e., the combination of single-target TCEs), indicating that CHT had in vitro specific killing activity, and the killing activity exhibited a "1+1>2" effect; CHT showed slightly greater in vitro activity than THC. In addition, the activity of CHT increased 50- to 100-fold relative to its controls, whereas the activity of THC increased only 7- to 12-fold relative to its controls, indicating that CHT was safer in vitro than THC. In addition, the high in vitro activity and safety of CHT were likewise demonstrated in A375 cells (FIG. 14B) and H460 cells (FIG. 14C). Example 13: Assessment of the Anti-Tumor Activity and Safety of the TDTCE Targeting EGFR and HER2 in a Mouse Xenograft Model Reconstituted with Activated Human T Cells (ATCs)

[0146] PBMCs were activated with 1 gg / mL anti-human CD3 antibody (purchased from BioLegend, Cat. No. 317326), 1 gg / mL anti-human CD28 antibody (purchased from BioLegend, Cat. No. 302934), and 300 IU IL-2 (purchased from Acnovia, Cat. No. AC52350), and the activated PBMC cells (called ATC) were obtained. 1.5 x 107 ATCs were injected via the tail vein into 6-week-old M-NSG female mice (purchased from Shanghai Model Organisms Center, Inc.), and simultaneously each mouse was subcutaneously administered IL-2 at 1000 IU / 100 pL. On the next day, 5 x 106 COLO205 cells were subcutaneously inoculated on the right axillary region of M-NSG mice, slightly toward the dorsal side, and the day of tumor inoculation was designated as Day 0. The mice were assigned to a PBS group, a CHT group, a CHG group, a GHT group, a THC group, a THG group, and a GHC group, with 6 mice per group. The corresponding treatments were administered respectively by tail-vein injection at 80 pgkg on Days 5 and 8, and each mouse simultaneously received 1,000 IU IL-2 in 100 pL by subcutaneous injection. Tumor growth was monitored during the study, tumor volume was calculated as length x width x width / 2, and mouse body weight was measured. When the mean tumor volume of the mice in the PBS group approached 1500 mm3, the mice were euthanized.

[0147] As shown in FIG. 15, CHT and THC exhibited potent tumor-killing activity in the ATC-reconstituted tumor-bearing mouse model, with a tumor inhibition rate of 100%. But the in vivo therapeutic efficacy of THG was similar to that of THC, indicating that THC had stronger killing activity against HER2-positive normal cells and a greater safety risk than CHT. In addition, no body-weight loss was observed in the CHT group, indicating that CHT had low toxicity and a favorable safety profile. Example 14: Safety Assessment of the TDTCE Targeting EGFR and HER2 in a Xenograft Model Using HER2-Transgenic Mice

[0148] The anti-human CD3 antibody HXR32 in CHT and THC was replaced with the antimouse CD3 antibody 145-2C11 (light-chain variable-region nucleotide sequence obtained from NCBI, accession no. U17870.1; heavy-chain variable-region nucleotide sequence obtained from NCBI, accession no. U17871.1). aEGFR-aCD3 / aHER2-2 (abbreviated as C1T; consisting of three subunits: CTX-H-hole-LALAPG-RF, CTX-L-145-2C11, whose gene sequence is as set forth in SEQ ID NO: 95, and Tra-scFv-knob) and T1C (consisting of three subunits: Tra-H-hole-LALAPG, whose gene sequence is the same as that in patent CN 118240093 A, Tra-L-145-2C11, whose gene sequence is as set forth in SEQ ID NO: 96, and CTX-scFv-knob, whose gene sequence is the same as that in patent CN 118240093 A) were constructed and expressed by the method described in Example 8.

[0149] Human HER2-transgenic mice aged 6-8 weeks (hHER2 mice; purchased from Shanghai Model Organisms Center, Inc.) were assigned to a PBS group, a C1T 500 pg / kg group, and a T1C 500 pg / kg group, with 5 mice per group. The corresponding treatment was administered by tail-vein injection every 4 days for a total of 4 doses. Mouse body weight was monitored during the study. At 24 h after the last dose, the mice were euthanized, and the heart, liver, lung, and kidney were collected for H&E staining analysis.

[0150] As shown in FIG. 16, no significant body-weight loss was observed in the C1T group, and no abnormalities were observed in the H&E histochemical staining results, indicating a favorable safety profile. In contrast, the body weight of the T1C group decreased significantly, and the H&E histochemical results suggested pulmonary toxicity, indicating that the drug posed a safety risk. Example 15: Assessment of the General Applicability of the aHER2 Sequence in aEGFR-aCD3 / aHER2

[0151] The anti-HER2 antibody was replaced with pertuzumab (abbreviated as Per; the sequence was obtained from NCBI, PDB: 1S78_D). CTX-H-hole-LALAPG-RF, CTX-L-HXR32, Per-scFv-knob (the gene sequence is as set forth in SEQ ID NO: 7), G12V-H-hole-LALAPG, and G12V-L-HXR32 were used to construct and express aEGFR-aCD3 / aHER2-3 (abbreviated as CHP) and its control molecule GHP by the method described in Example 8. COLO205 cells and PBMCs were seeded at a ratio of 1:4 in 96-well cell culture plates. Serial dilutions of CHP, GHP, and CHG were added (n = 3), mixed, and incubated at 37°C in a 5% CO2 incubator. After 48 h, the culture supernatant was collected and analyzed using an LDH detection kit, and the EC50 value was calculated utilizing GraphPad software.

[0152] As shown in FIG. 17, CHP was capable of mediating PBMC killing of COLO205 cells, and its activity was much greater than that of GHP and CHG, indicating that potent and specific anti-tumor activity could likewise be obtained by replacing the aHER2 sequence in aEGFR-aCD3 / aHER2. Example 16: Assessment of the General Applicability of the aEGFR Sequence in aEGFR-aCD3 / aHER2

[0153] The anti-EGFR antibody was replaced with panitumumab (abbreviated as Pan; its sequence was derived from patent US 6235883 B1). Pan-H-hole-LALAPG-RF (the gene sequence is as set forth in SEQ ID NO: 98), Pan-L-HXR32 (the gene sequence is as set forth in SEQ ID NO: 99), Tra-scFv-knob, and G12V-scFv-knob were used to construct and express aEGFR-aCD3 / aHER2-4 (abbreviated as PHT) and its control molecule PHG by the method described in Example 8. COLO205 cells and PBMCs were seeded at a ratio of 1:4 in 96-well cell culture plates. Serial dilutions of PHT, PHG, and GHT were added (n = 3), mixed, and incubated at 37°C in a 5% CO2 incubator. After 48 h, the culture supernatant was collected and analyzed using an LDH detection kit, and the EC50 value was calculated utilizing GraphPad software.

[0154] As shown in FIG. 18, PHT was capable of mediating PBMC killing of COLO205 cells, and this activity was far greater than that of PHG and GHT, indicating that potent and specific anti-tumor activity could likewise be obtained by replacing the aEGFR sequence in aEGFR-aCD3 / aHER2. Example 17: Assessment of the General Applicability of the aCD3 Sequence in aEGFR-aCD3 / aHER2

[0155] The anti-CD3 antibody was replaced with SP34 or I2C, and aEGFR-aCD3 / aHER2-5 (abbreviated as CST) and control molecules thereof GST and CSG, and aEGFR-aCD3 / aHER2-6 (abbreviated as CIT) and control molecules thereof GIT and CIG were constructed and expressed using CTX-H-hole-LALAPG-RF, CTX-L-SP34 (the gene sequence is as set forth in SEQ ID NO: 100), G12V-H-hole-LALAPG, G12V-L-SP34 (the gene sequence is as set forth in SEQ ID NO: 101), CTX-L-I2C (the gene sequence is as set forth in SEQ ID NO: 102), G12V-L-I2C (the gene sequence is as set forth in SEQ ID NO: 103), Tra-scFv-knob-, and G12V-scFv-knob (in the same manner as in Example 8). COLO205 cells and PBMCs were seeded at a ratio of 1:4 in 96-well cell culture plates. Serial dilutions of CST and its controls or CIT and its controls were added (n = 3), mixed, and incubated at 37°C in a 5% CO2 incubator. After 48 h, the culture supernatant was collected and analyzed using an LDH detection kit, and the EC50 value was calculated utilizing GraphPad software.

[0156] As shown in FIG. 19, CST and CIT were capable of mediating PBMC killing of COLO205 cells, and their activities were far greater than those of their respective controls, indicating that potent and specific anti-tumor activity could likewise be obtained by replacing the aCD3 sequence in aEGFR-aCD3 / aHER2. Example 18: Assessment of the Expression Level and In Vitro Activity of aEGFR-aCD3 / aHER2 after Hinge-Region Modification

[0157] The first cysteine (C) of the hinge region in the third subunit was mutated to serine (S), and CHT-3 (consisting of three subunits: CTX-H-hole-LALAPG-RF, CTX-L-HXR32, and Tra-scFv-S-knob (whose gene sequence is as set forth in SEQ ID NO: 104)), CHP-2 (consisting of three subunits: CTX-H-hole-LALAPG-RF, CTX-L-HXR32, and Per-scFv-S-knob (whose gene sequence is as set forth in SEQ ID NO: 105)), PHT-2 (consisting of three subunits: Pan-H-hole-LALAPG-RF, Pan-L-HXR32, and Tra-scFv-S-knob), CST-2 (consisting of three subunits: CTX-H-hole-LALAPG-RF, CTX-L-SP34, and Tra-scFv-S-knob), and CIT-2 (consisting of three subunits: CTX-H-hole-LALAPG-RF, CTX-L-I2C, and Tra-scFv-S-knob) were constructed and expressed in the same manner as in Example 8.

[0158] COLO205 cells and PBMCs were seeded at a ratio of 1:4 in 96-well cell culture plates. Serial dilutions of CHT, CHT-3, CHP, CHP-2, PHT, PHT-2, CST, CST-2, CIT, or CIT-2 were added (n = 3), mixed, and incubated at 37°C in a 5% CO2 incubator. After 48 h, the culture supernatant was collected and analyzed using an LDH detection kit, and the EC50 value was calculated utilizing GraphPad software.

[0159] As shown in Table 3 and FIG. 20, the expression level of aEGFR-aCD3 / aHER2 was significantly increased after hinge-region modification, while the modification had essentially no effect on the in vitro activity of aEGFR-aCD3 / aHER2.

[0160] Table 3 Expression level of aEGFR-aCD3 / aHER2 aEGFR-aCD3 / aHER2 Expression level (mg / L) CHT 95 CHT-3 121 CHP 92 CHP-2 109 PHT 87 PHT-2 102 CST 81 CST-2 92 CIT 74 CIT-2 89 Example 19: Assessment of the Expression Level and In Vitro Activity of aEGFR-aCD3 / aHER2 after Fc Modification

[0161] The RF mutation in the CH3 domain of the second subunit was replaced with an RC mutation, and CHT-4 (consisting of three subunits: CTX-H-hole-LALAPG-RC (whose gene sequence is as set forth in SEQ ID NO: 106), CTX-L-HXR32, and Tra-scFv-S-knob), CHP-3 (consisting of three subunits: CTX-H-hole-LALAPG-RC, CTX-L-HXR32, and Per-scFv-S-knob), PHT-3 (consisting of three subunits: Pan-H-hole-LALAPG-RC (whose gene sequence is as set forth in SEQ ID NO: 107), Pan-L-HXR32, and Tra-scFv-S-knob), CST-3 (consisting of three subunits: CTX-H-hole-LALAPG-RC, CTX-L-SP34, and Tra-scFv-S-knob), and CIT-3 (consisting of three subunits: CTX-H-hole-LALAPG-RC, CTX-L-I2C, and Tra-scFv-S-knob) were constructed and expressed in the same manner as in Example 8.

[0162] COLO205 cells and PBMCs were seeded at a ratio of 1:4 in 96-well cell culture plates. Serial dilutions of CHT-4, CHT-3, CHP-2, CHP-3, PHT-2, PHT-3, CST-2, CST-3, CIT-2, or CIT-3 were added (n = 3), mixed, and incubated at 37°C in a 5% CO2 incubator. After 48 h, the culture supernatant was collected and analyzed using an LDH detection kit, and the EC50 value was calculated utilizing GraphPad software.

[0163] Table 4 Expression level of aEGFR-aCD3 / aHER2 aEGFR-aCD3 / aHER2 Expression level (mg / L) CHT-4 120 CHT-3 121 CHP-3 112 CHP-2 109 PHT-3 98 PHT-2 102 CST-3 95 CST-2 92 CIT-3 91 CIT-2 89

[0164] As shown in Table 4 and FIG. 21, there was little difference in expression level or activity after the RC mutation or RF mutation was introduced into aEGFR-aCD3 / aHER2.

[0165] Although the specific embodiments of the present disclosure have been described above, it will be understood by those skilled in the art that these are merely illustrative, and that various alterations or modifications can be made to these embodiments without departing from the principle and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. A tumor dual-targeting T cell engager, wherein the tumor dual-targeting T cell engager comprises a first functional region, a second functional region, and a third functional region targeting EGFR, HER2, and CD3, respectively;the tumor dual-targeting T cell engager has the following structure:(1) the first functional region and the third functional region are scFv structures, the second functional region is a Fab structure, and the C-terminus of a light chain of the second functional region is connected to the N-terminus of the third functional region; or(2) the first functional region is a Fab structure, the second functional region and the third functional region are scFv structures, and the C-terminus of the light chain of the first functional region is connected to the N-terminus of the third functional region.

2. The tumor dual-targeting T cell engager of claim 1, wherein the scFv structure comprises a heavy-chain variable region (VH) and a light-chain variable region (VL) connected by a linker, and the scFv structure, from the N-terminus to the C-terminus, is: VL-linker-VH or VH-linker-VL; and / or, the Fab structure comprises a heavy-chain variable region (VH), a light-chain variable region (VL), a heavy-chain constant region 1 (CH1), and a light-chain constant region (CL), wherein the VH is connected to CH1, and the VL is connected to CL;preferably, the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 66-68; and / or, the CH1 and the CL are CH1 and CL of human IgG, respectively;more preferably, the CH1 has an amino acid sequence set forth in SEQ ID NO: 132; and / or, the CL has an amino acid sequence as set forth in SEQ ID NO: 65 or 133.

3. The tumor dual-targeting T cell engager of claim 1 or 2, wherein the tumor dualtargeting T cell engager further comprises an Fc, wherein the Fc is connected, by a hinge region, to the respective C-termini of the heavy-chain portions of the first functional region and the second functional region;preferably, the Fc has a knob-into-hole structure; and / or, the hinge region is a hinge region of human IgG;more preferably, the Fc comprises L234A / L235A / P329G mutations, and preferably 37further comprises H435R / Y436F mutations or H435R / Y436C mutations; and / or, the first cysteine (Cys) of the hinge region is substituted with serine (Ser).

4. The tumor dual-targeting T cell engager of any one of claims 1-3, wherein:the first functional region targeting EGFR comprises a VH and a VL, wherein (1) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; or (2) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, respectively;the second functional region targeting HER2 comprises a VH and a VL, wherein (i) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or (ii) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively;the third functional region targeting CD3 comprises a VH and a VL, wherein (a) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, respectively; or (b) the VH comprises HCDR1, HCDR2 and HCDR3, 38wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 43, SEQ ID NO: 117, and SEQ ID NO: 118, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 120, SEQ ID NO: 48, and SEQ ID NO: 121, respectively; or (c) the VH comprises HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 124, SEQ ID NO: 117, and SEQ ID NO: 125, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively;preferably:the first functional region targeting EGFR comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 37 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 37, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 41 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 41; or the first functional region targeting EGFR comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 111 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 111; and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 115 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 115;the first functional region targeting HER2 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 28 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 28, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 32 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 32; or the first functional region targeting HER2 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 58 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 58, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 62 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 62;the first functional region targeting CD3 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 46 or is an amino acid sequence having at 39least 80% sequence identity to SEQ ID NO: 46, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 50 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 50; or the first functional region targeting CD3 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 119 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 119, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 122 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 122; or the first functional region targeting CD3 comprises a VH, wherein the amino acid sequence of the VH is as set forth in SEQ ID NO: 126 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 126, and a VL, wherein the amino acid sequence of the VL is as set forth in SEQ ID NO: 130 or is an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 130;more preferably:when the first functional region is the scFv structure, the first functional region has an amino acid sequence as set forth in SEQ ID NO: 42 or 116 or has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 42 or 116;when the second functional region is the scFv structure, the first functional region has an amino acid sequence as set forth in SEQ ID NO: 33 or 63 or has an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 33 or 63;when the third functional region is the scFv structure, the first functional region has an amino acid sequence as set forth in any one of SEQ ID NOs: 51, 123, and 131 or has an amino acid sequence with at least 80% sequence identity to any one of SEQ ID NOs: 51, 123, and 131.

5. The tumor dual-targeting T cell engager of any one of claims 1-4, wherein the tumor dual-targeting T cell engager comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain;the structure of the first polypeptide chain, from the N-terminus to the C-terminus, is: VL-CL-scFv1;the structure of the second polypeptide chain, from the N-terminus to the C-terminus, is: 40VH-CH1-Fc1;the structure of the third polypeptide chain, from the N-terminus to the C-terminus, is: scFv2-Fc2;the VH and VL, scFv1, and scFv2 target different antigens, wherein the antigens are selected from EGFR, HER2, and CD3; Fc1 and Fc2 are identical or form a knob-into-hole structure;preferably, the structure of the scFv1, from the N-terminus to the C-terminus, is: VL1-linker-VH1, and the structure of the scFv2, from the N-terminus to the C-terminus, is: VL2-linker-VH2.

6. The tumor dual-targeting T cell engager of claim 5, wherein the VH and VL target EGFR or HER2, the scFv1 targets CD3, and the scFv2 targets HER2 or EGFR;preferably, the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from any one of the following structures:(1) the first polypeptide chain: EGFR VL-CL-CD3 scFv; the second polypeptide chain: EGFR VH-CH1-Fc1; the third polypeptide chain: HER2 scFv-Fc2;(2) the first polypeptide chain: HER2 VL-CL-CD3 scFv; the second polypeptide chain: HER2 VH-CH1-Fc1; the third polypeptide chain: EGFR scFv-Fc2;more preferably:the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 2; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 1; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 3; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the firstpolypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the secondpolypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 134; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the firstpolypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the secondpolypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third 41polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 87; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84;or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 79; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 78; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 79; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 78; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 79; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 90; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84;or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 9; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 8; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 3; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 77; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 71; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 86;or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 80; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 80; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 80; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 87; the third 42polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84;or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 82; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 72; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 82; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 70; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84; or the first polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 82; the second polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 87; the third polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 84.

7. A polynucleotide, wherein the polynucleotide encodes the tumor dual-targeting T cell engager of any one of claims 1-6;preferably, the polynucleotide comprises a first nucleic acid, a second nucleic acid, and a third nucleic acid; the first nucleic acid, the second nucleic acid, and the third nucleic acid encode the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain, respectively;more preferably, the first nucleic acid has a sequence as set forth in any one of SEQ ID NOs: 12, 19, 92, 99, 100, and 102; the second nucleic acid has a sequence as set forth in any one of SEQ ID NOs: 11, 18, 91, 98, 106, 107, and 135; the third nucleic acid has a sequence as set forth in any one of SEQ ID NOs: 13, 93, 97, 104, and 105.

8. A recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide of claim 7.

9. A transformant, wherein the transformant expresses the tumor dual-targeting T cell engager of any one of claims 1-6, or comprises the polynucleotide of claim 7, or comprises the recombinant expression vector of claim 8.

10. A method for preparing a tumor dual-targeting T cell engager, wherein the method 43comprises culturing the transformant of claim 9, and obtaining the tumor dual-targeting T cell engager from the culture.

11. A pharmaceutical composition, wherein the pharmaceutical composition comprises the tumor dual-targeting T cell engager of any one of claims 1-6, the polynucleotide of claim 7, the recombinant expression vector of claim 8, and / or the transformant of claim 9, and optionally a pharmaceutically acceptable carrier and / or excipient.

12. Use of the tumor dual-targeting T cell engager of any one of claims 1-6, the polynucleotide of claim 7, the recombinant expression vector of claim 8, the transformant of claim 9, or the pharmaceutical composition of claim 11 in the manufacture of a medicament for treating cancer;preferably, the cancer is selected from breast cancer, gastric cancer, non-small cell lung cancer, colon cancer, epidermoid carcinoma, and melanoma.

13. An anti-tumor drug, wherein the anti-tumor drug comprises the tumor dual-targeting T cell engager of any one of claims 1-6, the polynucleotide of claim 7, or the pharmaceutical composition of claim 11 as an active ingredient.

14. The tumor dual-targeting T cell engager of any one of claims 1-6, the polynucleotide of claim 7, the recombinant expression vector of claim 8, the transformant of claim 9, the pharmaceutical composition of claim 11, or the anti-tumor drug of claim 13, for use in treating cancer;preferably, the cancer is selected from breast cancer, gastric cancer, non-small cell lung cancer, colon cancer, epidermoid carcinoma, and melanoma.

15. A method for treating cancer, wherein the method comprises administering to a subject in need thereof an effective amount of the tumor dual-targeting T cell engager of any one of claims 1-6, the polynucleotide of claim 7, the recombinant expression vector of claim 8, the transformant of claim 9, the pharmaceutical composition of claim 11, or the anti-tumor drug of 44claim 13;preferably, the cancer is selected from breast cancer, gastric cancer, non-small cell lung cancer, colon cancer, epidermoid carcinoma, and melanoma.45

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