Three-specificity T cell adapter targeting HER2, HER3 and CD3 as well as preparation method and application of three-specificity T cell adapter

Abstract

The invention relates to a three-specificity T cell adapter targeting HER2 (human epidermal growth factor receptor 2), HER3 (human epidermal growth factor receptor 3) and CD3 (human epidermal growth factor receptor 3) as well as a preparation method and application thereof. The three-specificity T cell adapter comprises an HER2 antigen binding structural domain, an HER3 antigen binding structural domain and a CD3 antigen binding structural domain which respectively target a human HER2 protein, a human HER3 protein and a human CD3 protein; the HER3 binding structural domain and the HER2 binding structural domain can target tumor cells expressing the two antigens, the CD3 binding structural domain can target T cells, the three-specificity T cell adapter can shorten the distance between the tumor cells and the T cells, and the T cells are stimulated to activate, so that the T cells have cytotoxic effect on the tumor cells; by combining the anti-HER3 binding structural domain and the anti-HER2 binding structural domain, on one hand, the action range and the action effect of the three-specificity T cell adapter can be increased, and on the other hand, the purposes of increasing specificity and reducing non-specific toxic and side effects can be achieved by targeting two different antigens of the same tumor cell at the same time.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering antibody technology, and in particular relates to a trispecific T cell conjugate targeting HER2, HER3 and CD3, its preparation method and application. Background Technology

[0002] T cells can kill tumor cells, but because the expression of MHC on the surface of most tumor cells is often downregulated or even absent, tumor cells can evade T cell surveillance and killing through immune escape. At the same time, tumor cells highly express immune checkpoint ligand molecules (such as PD-L1), which inhibit T cell activity by binding to immune checkpoint receptors on T cells.

[0003] T cell engagers (TCEs) are bispecific antibodies that simultaneously bind to tumor-associated antigens (TAAs) and T cells, with the T cell binding site being CD3ε in the T cell receptor complex. TCEs can shorten the distance between tumor cells and T cells, mediating the formation of an immune synapse, thereby stimulating T cells to release perforin and granzyme to kill tumor cells. T cell engagers composed of fragmented antibodies (single-chain antibodies, nanobodies, etc.) have the characteristics of small molecular weight, flexible structure, and high tissue permeability. Compared with IgG-formatted TCEs, they can act rapidly on lesion sites and increase the infiltration of immune cells in the tumor microenvironment. Existing T cell engagers suffer from side effects such as cytokine storm syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), which severely limit their application scenarios. Furthermore, due to insufficient specificity, off-target toxicity caused by killing non-tumor cells is also a problem that urgently needs to be addressed. Therefore, its geometry, affinity, and valence state need to be finely adjusted to balance efficacy and safety. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a trispecific T-cell conjugate targeting HER2, HER3, and CD3, along with its preparation method and applications.

[0005] The technical solution adopted in this invention is: a trispecific T cell binder targeting HER2, HER3 and CD3, comprising a HER2 antigen-binding domain, a HER3 antigen-binding domain and a CD3 antigen-binding domain.

[0006] Preferably, the HER2 antigen-binding domain, HER3 antigen-binding domain, and CD3 antigen-binding domain include a heavy chain variable region and a light chain variable region. The heavy chain variable region and light chain variable region of the CD3 antigen-binding domain are cross-connected with the heavy chain variable region and light chain variable region of the HER2 / HER3 antigen-binding domain, and then connected upstream to the heavy chain variable region and light chain variable region of another HER3 / HER2 antigen-binding domain.

[0007] Preferably, the HER2 antigen-binding domain comprises a heavy chain variable region HER2 VH and a light chain variable region HER2 VL; HER2 VH comprises one or more of the amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID No. 20, SEQ ID No. 22, and SEQ ID No. 24, respectively, and HER2 VL comprises one or more of the amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID No. 14, SEQ ID No. 16, and SEQ ID No. 18, respectively;

[0008] The HER3 antigen-binding domain comprises a heavy chain variable region HER3 VH and a light chain variable region HER3 VL; HER3 VH comprises one or more of the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID No. 32, SEQ ID No. 34, and SEQ ID No. 36, respectively; HER3 VL comprises one or more of the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID No. 26, SEQ ID No. 28, and SEQ ID No. 30, respectively.

[0009] The CD3 antigen-binding domain comprises a heavy chain variable region CD3 VH and a light chain variable region CD3 VL; CD3 VH comprises one or more of the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID No. 44, SEQ ID No. 46, and SEQ ID No. 48, respectively; and CD3 VL comprises one or more of the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID No. 38, SEQ ID No. 40, and SEQ ID No. 42, respectively.

[0010] Preferably, the HER2 antigen-binding domain includes a heavy chain variable region as shown in SEQ ID No. 3 and a light chain variable region as shown in SEQ ID No. 1; the HER3 antigen-binding domain includes a heavy chain variable region HER3 VH as shown in SEQ ID No. 7 and a light chain variable region HER3 VL as shown in SEQ ID No. 5; and the CD3 antigen-binding domain includes a heavy chain variable region CD3 VH as shown in SEQ ID No. 11 and a light chain variable region CD3 VL as shown in SEQ ID No. 9.

[0011] Preferably, the heavy chain variable region and light / heavy chain variable region of each antigen-binding domain in the trispecific T cell conjugate are connected by a linker peptide. The linker peptide sequences at different positions may be the same, partially the same, or different, and the linker peptide sequences are as shown in any of SEQ ID No. 49-52.

[0012] Preferably, the trispecific T-cell binder comprises, in sequence, a first functional region, a second functional region, a third functional region, a fourth functional region, a fifth functional region, and a sixth functional region; each functional region is connected by a linker peptide;

[0013] The first and second functional areas each include either VH_HER3 or VL_HER3; the third, fourth, fifth, and sixth functional areas each include one of VL_HER2, VH_CD3, VH_HER2, and VL_CD3, wherein VL_HER2 and VH_HER2 are not directly connected, and VH_CD3 and VL_CD3 are not directly connected.

[0014] Alternatively, the first and second functional areas may each include VH_HER2 or VL_HER2; the third, fourth, fifth, and sixth functional areas may each include one of VL_HER3, VH_CD3, VH_HER3, and VL_CD3, wherein VL_HER3 and VH_HER3 are not directly connected, and VH_CD3 and VL_CD3 are not directly connected.

[0015] Preferably, the first functional region and the second functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 49; the second functional region and the third functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 50; the third functional region and the fourth functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 51; the fourth functional region and the fifth functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 52; and the fifth functional region and the sixth functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 51.

[0016] A method for preparing a trispecific T-cell conjugate targeting HER2, HER3, and CD3 was developed. An expression vector capable of expressing the trispecific T-cell conjugate was constructed, the expression vector was transfected into host cells, and after culture, the expression product was separated and purified to obtain a protein solution containing the trispecific T-cell conjugate.

[0017] Preferably, when constructing the expression vector, histidine tags and Strep tag-II tags are added to the N-terminus and C-terminus of the gene fragment expressing the trispecific T cell binder, respectively; the expression vector is pET28a(−), and the host cell is the Escherichia coli prokaryotic expression system.

[0018] Application of trispecific T-cell binders targeting HER2, HER3, and CD3 in drugs for treating tumors or related diseases caused by HER2-specific antigen expression.

[0019] Preferably, the tumor may be breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, and soft tissue cancer; preferably, the tumor is selected from breast cancer and gastric cancer.

[0020] The advantages and positive effects of this invention are as follows: the HER3-binding domain and HER2-binding domain in the trispecific T-cell conjugate can target tumor cells expressing these two antigens, while the CD3-binding domain can target T cells. Through the action of the trispecific T-cell conjugate, the distance between tumor cells and T cells is shortened, stimulating T-cell activation and enabling them to produce cytotoxic effects on tumor cells. The combination of the anti-HER3 binding domain and the anti-HER2 binding domain can also increase specificity and reduce non-specific toxic side effects by simultaneously targeting two different antigens of the same tumor cell.

[0021] The CD3 antigen-binding domain and the HER2 / HER3 antigen-binding domain are arranged in a DART format, and the connecting peptide structure between each domain is further defined, which facilitates the capture of tumor cells in the environment and helps to stabilize the formation of a stable immune synapse between T cells and tumor cells, thereby continuously killing tumor cells.

[0022] The trispecific T-cell conjugate has a molecular weight of approximately 83 kDa, about half that of IgG-like antibodies, and exhibits better tissue penetration compared to traditional monoclonal antibodies. It also has a longer plasma half-life compared to nanobodies, single-chain antibodies, and Fab-form antibodies. Furthermore, as a single-chain fusion protein, the trispecific T-cell conjugate avoids the heavy and light chain mismatch issues associated with IgG-like antibodies, resulting in stable downstream production processes, simple and efficient purification steps, and uniform expression products. Attached Figure Description

[0023] Figure 1 Schematic diagram of the structure of the three-specific T cell conjugate;

[0024] Figure 2 Atlas of expression vector plasmids containing trispecific T cell conjugates;

[0025] Figure 3 SDS-PAGE results of purification of the trispecific T cell conjugate under denaturing conditions;

[0026] Figure 4 SDS-PAGE results of triple specific T cell conjugates purified by secondary affinity using Streptactin-XT packing material;

[0027] Figure 5 Indirect ELISA was used to test the binding ability of the trispecific T cell binder to target proteins; A: binding ability with HER2 protein; B: binding ability with HER3 protein; C: binding ability with CD3 protein.

[0028] Figure 6 Flow cytometry was used to examine the binding ability of the trispecific T cell binder to target cells.

[0029] Figure 7 Western blot assays were used to examine the ability of the trispecific T cell conjugate to mediate the downregulation of Akt protein phosphorylation in SK-BR-3 cells.

[0030] Figure 8 Western blot assays were used to examine the ability of the trispecific T cell conjugate to mediate downregulation of Akt protein phosphorylation in NCI-N87 cells.

[0031] Figure 9 The effects of CCK-8 assay on the proliferation of target cells by the trispecific T cell conjugate; A: Effect on the proliferation of SK-BR-3; B: Effect on the proliferation of NCI-N87; C: Effect on the proliferation of MDA-MB-453;

[0032] Figure 10 Immunofluorescence assay to detect the ability of antibodies to mediate immune synapse formation;

[0033] Figure 11 Flow cytometry was used to detect the ability of antibodies to activate CD69, an early marker of T cell activation.

[0034] Figure 12 Flow cytometry was used to detect the ability of antibodies to activate CD25, a mid-stage marker of T-cell activation.

[0035] Figure 13CCK-8 assay detects the ability of antibody-mediated T cell killing of target cells; A: T cell killing ability of SK-BR-3 cells; B: T cell killing ability of NCI-N87 cells. Detailed Implementation

[0036] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0037] This invention relates to a trispecific T-cell conjugate targeting HER2, HER3, and CD3, its preparation method, and its applications. The trispecific T-cell conjugate includes a HER2 antigen-binding domain, a HER3 antigen-binding domain, and a CD3 antigen-binding domain, targeting human HER2, human HER3, and human CD3 proteins, respectively. The HER3 and HER2 binding domains can target tumor cells expressing these two antigens, while the CD3 binding domain targets T cells. The trispecific T-cell conjugate brings tumor cells and T cells closer together, stimulating T-cell activation and enabling them to exert cytotoxic effects on tumor cells. The combination of the anti-HER3 and anti-HER2 binding domains increases the range and efficacy of the trispecific T-cell conjugate. Furthermore, by simultaneously targeting two different antigens of the same tumor cell type, it increases specificity and reduces non-specific toxicity.

[0038] HER2 and HER3 both belong to the epidermal growth factor receptor (EGFR) family. This family consists of four homologous receptor tyrosine kinases (RTKs): HER1 (EGFR), HER2, HER3 (ErbB3), and HER4 (ErbB4). They share similar basic structures: an extracellular ligand-binding domain, an α-helical transmembrane domain, a cytoplasmic tyrosine kinase domain (HER3 lacks or has minimal tyrosine kinase activity), and a C-terminal signaling domain. The HER2 gene is widely distributed in breast cancer (20%–30%). Like other EGFR family members, its extracellular domain consists of four subdomains, from N-terminus to C-terminus: Domain I (L1), II (CR1), III (L2), and IV (CR2). L1 and L2 are leucine-rich repeats (LRRs) that can directly interact with ligands; CR1 and CR2 are cysteine-rich and responsible for disulfide bond formation during dimerization. The crystal structure of HER2 shows that its ligand-binding site is closed, with extracellular domains I and III in direct contact; therefore, HER2 does not yet have a known natural ligand. In its open conformation, HER2 is similar to other HER2 molecules after ligand activation, and can directly form HER2 dimers or heterodimers with other EGFR members. The primary ligand for HER3 is neuroregulatory protein 1 (NRG1). In the absence of a ligand, extracellular domains II and IV interact directly, resulting in an inactive "closed" conformation for HER3. With a ligand present, it binds between domains I and III, causing HER3 to transition to an "open" conformation, exposing the dimerization site located in domain II. Subsequently, HER3 can form heterodimers with other EGFR family members (primarily HER2).

[0039] HER2 is the most extensively studied drug target in the EGFR family, with trastuzumab and pertuzumab already approved for clinical treatment, and several other monoclonal antibodies and multispecific antibodies in clinical development. While there are no marketed monoclonal antibody drugs targeting HER3, it has been shown to be associated with resistance to small molecule drugs such as gefitinib, lapatinib, and trastuzumab. HER2 / HER3 is the most potent EGFR dimer and is the most common in breast cancer, representing one of the mechanisms driving tumor drug resistance. Therefore, restricting the binding of HER3 to its ligands and the formation of HER2 homodimers and heterodimers are important research directions for improving the treatment outcomes of breast cancer.

[0040] In the trispecific T-cell binder, the HER2 antigen-binding domain, HER3 antigen-binding domain, and CD3 antigen-binding domain all include heavy chain variable regions and light chain variable regions. The heavy chain and light chain variable regions of the CD3 antigen-binding domain are cross-connected with the heavy chain and light chain variable regions of the HER2 / HER3 antigen-binding domain, arranged in a DART format, and then upstream, connected to the heavy chain and light chain variable regions of another HER3 / HER2 antigen-binding domain. For example, the variable regions of the CD3 antibody and HER2 antibody are arranged in a DART format, and then upstream, the variable region of the HER3 antibody is connected; or the variable regions of the CD3 antibody and HER3 antibody are arranged in a DART format, and then upstream, the variable region of the HER2 antibody is connected.

[0041] In some embodiments of the present invention, the HER2 antigen-binding domain comprises a heavy chain variable region (HER2 VH) and a light chain variable region (HER2 VL); the HER2 VH comprises one or more of the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID No. 20, SEQ ID No. 22, and SEQ ID No. 24, respectively, and the HER2 VL comprises one or more of the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID No. 14, SEQ ID No. 16, and SEQ ID No. 18, respectively. The HER3 antigen-binding domain comprises a heavy chain variable region (HER3 VH) and a light chain variable region (HER3 VL); the HER3 VH comprises one or more of the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID No. 32, SEQ ID No. 34, and SEQ ID No. 36, respectively, and the HER3 VL comprises one or more of the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID No. 26, SEQ ID No. 28, and SEQ ID No. 30, respectively. The CD3 antigen-binding domain comprises a heavy chain variable region (CD3 VH) and a light chain variable region (CD3 VL); the CD3 VH comprises one or more of the amino acid sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID No. 44, SEQ ID No. 46 and SEQ ID No. 48, respectively, and the CD3 VL comprises one or more of the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID No. 38, SEQ ID No. 40 and SEQ ID No. 42, respectively.

[0042] In some embodiments of the present invention, the parent antibody for the HER3 binding domain of the trispecific T-cell conjugate is seribantumab, which targets HER3 extracellular domain I; the parent antibody for the HER2 binding domain is pertuzumab, which targets HER2 extracellular domain II. This pairing of antigen-binding domains helps to inhibit the production of HER2 / HER2 dimers and HER2 / HER3 heterodimers, further inhibiting the transduction of downstream signaling pathways and thus inhibiting the proliferation and metastasis of tumor cells.

[0043] The HER2 antigen-binding domain comprises a heavy chain variable region (HER2 VH) and a light chain variable region (HER2 VL); the amino acid sequence of HER2 VL is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 2; the amino acid sequence of HER2 VH is shown in SEQ ID No. 3, and the nucleotide sequence is shown in SEQ ID No. 4. The HER3 antigen-binding domain comprises a heavy chain variable region (HER3 VH) and a light chain variable region (HER3 VL); the amino acid sequence of HER3 VL is shown in SEQ ID No. 5, and the nucleotide sequence is shown in SEQ ID No. 6; the amino acid sequence of HER3 VH is shown in SEQ ID No. 7, and the nucleotide sequence is shown in SEQ ID No. 8. The CD3 antigen-binding domain comprises a heavy chain variable region (CD3 VH) and a light chain variable region (CD3 VL); the amino acid sequence of CD3 VL is shown in SEQ ID No. 9, and the nucleotide sequence is shown in SEQ ID No. 10; the amino acid sequence of CD3 VH is shown in SEQ ID No. 11, and the nucleotide sequence is shown in SEQ ID No. 12.

[0044] Table 1

[0045]

[0046] In the trispecific T-cell conjugate, the heavy chain variable region and the light chain variable region of each antigen-binding domain are connected by linker peptides. The linker peptide sequences at different positions may be the same, partially the same or different, and the linker peptide sequences are shown in any of SEQ ID No. 49-52.

[0047] Table 2

[0048]

[0049] The trispecific T-cell binder comprises, sequentially, a first functional region, a second functional region, a third functional region, a fourth functional region, a fifth functional region, and a sixth functional region; each functional region is connected by a linker peptide. Specifically, the first and second functional regions each contain either VH_HER3 or VL_HER3, which are connected via a Linker (G4S)3 linker peptide; the third, fourth, fifth, and sixth functional regions each contain one of VL_HER2, VH_CD3, VH_HER2, and VL_CD3, wherein VL_HER2 and VH_HER2 are not directly connected, and VH_CD3 and VL_CD3 are not directly connected. Alternatively, the first and second functional regions may each include VH_HER2 or VL_HER2, which are linked by a Linker (G4S)3 linker peptide; the third, fourth, fifth, and sixth functional regions may each include one of VL_HER3, VH_CD3, VH_HER3, and VL_CD3, wherein VL_HER3 and VH_HER3 are not directly linked, and VH_CD3 and VL_CD3 are not directly linked.

[0050] The second and third functional regions are linked via a linker G4S linker peptide; the third and fourth functional regions are linked via a linker G3SG4 linker peptide; the fourth and fifth functional regions are linked via a linker-SS linker peptide; and the fifth and sixth functional regions are linked via a linker G3SG4 linker peptide. In some embodiments of the present invention, the antibody fragments in the trispecific T-cell conjugate can be linked in the following order: VH_HER3-VL_HER3-VL_HER2-VH_CD3-VH_HER2-VL_CD3, or VH_HER3-VL_HER3-VL_HER2-VL_CD3-VH_HER2-VH_CD3, or VH_HER2-VL_HER2-VL_HER3-VH_CD3-VH_HER3-VL_CD3, or VH_HER2-VL_HER2-VL_HER3-VL_CD3-VH_HER3-VH_CD3.

[0051] The CD3 VL and HER2 VH are linked by a linker peptide with the amino acid sequence VEPKSCGGGSCEGRNF, introducing a disulfide bond to stabilize the structure and help stabilize the formation of a stable immune synapse between T cells and tumor cells, thus continuously killing tumor cells. The HER3 binding domain and the HER2-CD3 domain are linked by a short linker peptide with the amino acid sequence GGGGS, maintaining a flexible structure between the HER3 binding domain and the HER2 binding domain, which helps the trispecific T cell binder capture tumor cells in the environment.

[0052] To prepare a trispecific T-cell conjugate, a gene fragment containing HER2, HER3, and CD3 antigen-binding domains was first constructed into an expression vector. The expression vector plasmid was then transfected into host cells. After cell culture, the target antibody was expressed. The expression product was extracted and purified to obtain a protein solution suitable for use as a trispecific T-cell conjugate. The expression vector can be pET28a(−), and the host cell can be an *E. coli* prokaryotic expression system. To facilitate purification, histidine tags and Strep tag-II tags were added to the N-terminus and C-terminus of the designed gene fragment containing HER2, HER3, and CD3 antigen-binding domains, respectively, for affinity purification.

[0053] Based on the specific structure of the trispecific T-cell binder, by targeting HER2 / HER3-expressing tumor cells and T cells, the distance between tumor cells and T cells can be shortened, stimulating T-cell activation and enabling them to exert cytotoxic effects on tumor cells. This can be used as a drug to treat tumors or related diseases caused by HER2 and / or HER3 specific antigen expression, or to kill cells expressing HER2 and / or HER3. In some embodiments of the present invention, the tumor may be breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, and soft tissue cancer; preferably, the tumor is selected from breast cancer and gastric cancer.

[0054] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0055] Example 1: Preparation of a Trispecific T-cell binder

[0056] 1.1 Design of Trispecific T-cell binders

[0057] First, a trispecific T-cell binder was designed and constructed, in which the order of each antigen domain is VH_HER3, VL_HER3, VL_HER2, VH_CD3, VH_HER2, and VL_CD3.

[0058] Pertuzumab was selected as the parental HER2 antibody, with sequences shown in SEQ ID No. 2 and SEQ ID No. 4. Pertuzumab targets the extracellular domain II of the HER2 protein, close to its dimerization site, and therefore can better block HER2 dimerization compared to Trastuzumab. Seribantumab, which targets the extracellular domain I of HER3 and can competitively bind to HER3 with NRG1, was selected as the parental HER3 antibody, with sequences shown in SEQ ID No. 6 and SEQ ID No. 8. It inhibits HER3 activation at the source to suppress the formation of the HER2 / HER3 complex. TR66 antibody, which is the same as Blinatumomab, was selected as the parental antibody targeting CD3, with sequences shown in SEQ ID No. 10 and SEQ ID No. 12.

[0059] The trispecific T-cell binder format is designed based on the BiTE and DART formats. The BiTE format consists of two scFvs linked together by a GS Linker, which has high flexibility and facilitates antibody capture of cells from multiple angles. Although the DART format is also composed of two pairs of antibody variable regions, the introduction of interchain disulfide bonds not only stabilizes the antibody structure but also makes the distance between the two variable regions relatively fixed and spatially opposite, which is conducive to the formation of immune synapses between tumor cells and T cells.

[0060] The variable regions of the CD3 and HER2 antibodies were arranged in a DART format; simultaneously, GSLinker was used to link the variable regions of the HER2 and HER3 antibodies, thus forming a BiTE-like format. To avoid mispairing or failure of the two antibody chains to pair, short GS peptides were added at disulfide bond introduction sites to link the two DART chains, ensuring that the entire antibody molecule consists of only a single chain, as shown in the structure. Figure 1 As shown. Finally, histidine tags and Strep tag-II tags were added to the N-terminus and C-terminus, respectively, for affinity purification.

[0061] 1.2 Construction of expression vector

[0062] Based on the design of the aforementioned trispecific T-cell binder, Beijing Qingke Biotechnology Co., Ltd. was commissioned to construct the nucleic acid sequence of the trispecific T-cell binder and then constructed it into the pET28a(−) plasmid. The plasmid structure is shown below. Figure 2 As shown.

[0063] 1.3 Prokaryotic expression and purification of the trispecific T cell conjugate

[0064] The constructed pET28a(−)-START plasmid was transformed into BL21(DE3) engineered bacteria to obtain a prokaryotic expression engineered bacteria for the trispecific T cell conjugate.

[0065] The engineered bacteria were cultured and induced to express the trispecific T cell conjugate on a large scale. A 1 L Erlenmeyer flask was filled with 600 mL of LB medium containing kanamycin and 6 mL of the engineered bacterial seed culture. The mixture was incubated at 37°C with shaking at 200 rpm for approximately 3 hours until the logarithmic growth phase was reached. 300 μL of IPTG solution was then added, and the culture was continued for another 6 hours. The induced bacterial culture was centrifuged at 4°C and 8500 rpm for 15 min using a high-speed refrigerated centrifuge to collect the bacterial cells and weigh them. The cells were resuspended in bacterial suspension at a ratio of 1:15 (w / v). The cells were then sonicated on ice (390 W, 3 s on, 8 s off, total 10 min). After centrifugation at 12,000 rpm for 20 min, inclusion body precipitate was obtained. The inclusion bodies were resuspended sequentially with inclusion body washing solutions I, II, and III, and then with PBS solution. The mixture was washed for 1 hour with stirring, centrifuged at 12,000 rpm for 20 min, and the precipitate was collected and washed again. This process was repeated four times in total. After washing, the inclusion bodies were resuspended in 20 mL of a one-step denaturing solution containing 8 M urea. The solution was stirred at room temperature for 30 min and then incubated overnight at 4°C. The next day, the bodies were removed and centrifuged at 12,000 rpm for 20 min. The supernatant was filtered through a 0.22 μm microporous membrane. A first affinity purification was performed using Ni-NTA packing material under denaturing conditions. The purification results are shown in [Figure number missing]. Figure 3 Collect the purified protein solution and dialyze it into a denaturing buffer. Perform secondary affinity purification using Streptactin-XT packing material. The purification results are shown in [Figure number missing]. Figure 4 A purified trispecific T cell binder protein solution was obtained. The purified protein was dialyzed to remove salt and concentrated, and then stored at -80°C.

[0066] Example 2: Performance Analysis of the Trispecific T-cell Connector

[0067] 2.1 Indirect ELISA method for detecting the affinity of the trispecific T cell binder for target proteins

[0068] The affinity of the trispecific T cell conjugates prepared in Example 1 for HER2, HER3, and CD3 proteins was detected by indirect ELISA. The specific steps are as follows: (1) Antigen coating: The antigens to be coated were diluted to specific concentrations (HER2, CD3: 0.5 μg / mL; HER3: 1 μg / mL) using antigen coating solution, added to a 96-well plate (100 μL / well), and coated overnight at 4°C (at least 12 h). (2) Blocking: The liquid in the wells was poured out, 200 μL of PBST was added to each well, and the plate was washed for 15 s on a shaker at 170 rpm. The washing solution was then discarded, and the process was repeated three times. The residual liquid was blotted off on absorbent paper, and 200 μL of blocking solution was added to each well. The plate was then placed in a constant temperature shaker at 37°C for 2 h. (3) Primary antibody incubation: After blocking, the plate was washed three times and the residual liquid was blotted off. 100 μL of serially diluted antibody was added to each well, and the plate was reacted at 37°C for 1.5 h. (4) Secondary antibody incubation: After the primary antibody incubation, wash five times and remove residual liquid. Add 100 μL of Strep-Tag II assay reagent (PBST 1:5000 dilution) coupled with HRP to each well and react at 37°C for 1.5 h. (5) HRP color development: After the secondary antibody incubation, wash five times and remove residual liquid. Add 100 μL of soluble TMB single component to each well and let stand at room temperature for 15 min. Avoid light during this step. (6) Termination of color development: Add 50 μL of ELISA stop solution to each well and gently tap to mix the system. (7) Detection: Detect the absorbance at a wavelength of 450 nm using a microplate reader, record the data, and use GraphpadPrism software to perform four-parameter curve fitting.

[0069] The results are as follows Figure 5 As shown, the trispecific T-cell binder can effectively bind HER2, HER3, and CD3 proteins.

[0070] 2.2 Flow cytometry detection of the binding ability of trispecific T cell binders to target cells

[0071] The binding ability of the trispecific T cell conjugate prepared in Example 1 to target cells was tested using NCI-N87, SK-BR-3, MDA-MB-453, OVCAR-3, MCF-7, FaDu, HCT116 and Jurkat cells as target cells, respectively.

[0072] Digest and count the cells, then transfer them to 1.5 mL centrifuge tubes, so that each tube contains 2 × 10⁶ cells. 5Cells were washed twice with 200 μL of pre-chilled flow cytometry buffer, centrifuged at 600×g for 5 min at 4°C. Cells were resuspended in 100 μL of antibody and incubated at 4°C for 1.5 h. Cells were washed twice, and then resuspended in 100 μL of 1:5000 diluted Strep-tagged monoclonal antibody and incubated at 4°C for 1.5 h. Cells were washed twice, and then resuspended in 1:100 diluted FITC-labeled goat anti-mouse IgG and incubated at 4°C for 0.5 h. Light should be avoided during this process. Cells were washed twice, resuspended in 500 μL of flow cytometry buffer, and then analyzed.

[0073] The results are as follows Figure 6 As shown, the trispecific T cell binder can effectively bind to cells such as NCI-N87, SK-BR-3, MDA-MB-453, OVCAR-3, MCF-7, FaDu, HCT116, and Jurkat.

[0074] 2.3 Western Blot analysis of the effect of the trispecific T cell binder on the PI3K-Akt signaling pathway

[0075] Using SK-BR-3 and NCI-N87 cells as models, the effect of the trispecific T cell binder on the phosphorylation of Akt protein in the two cell types was investigated.

[0076] Digest and count the cells, transfer them to 12-well plates, and add 1-2 × 10⁶ cells per well. 5Cells were collected. When the cell confluence reached 80%, the culture medium was replaced with serum-free medium and starved for 24 h. The cells were washed once with PBS, and 500 μL of serum-free medium containing different concentrations of antibodies was added, and the cells were cultured for another 3 h. NRG protein was added to a final concentration of 10 ng / mL and incubated for 0.5 h. Sample preparation: The cell plate was placed on ice and washed twice with pre-chilled PBS buffer. 60 μL of RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added to each well, and the cells were lysed on ice for 20 min. Cells were then scraped from top to bottom with a cell scraper, and the cell suspension was transferred to a pre-chilled 1.5 mL centrifuge tube. The cells were repeatedly pipetted 50 times to ensure complete lysis. The cells were centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was collected and denatured at 55°C for 15 min with Loading Buffer. Electrophoresis: SDS-PAGE was performed without Coomassie Brilliant Blue staining. Note that 1× Loading Buffer should be added to each sample well to fill the gap, and the same volume of 1× Loading Buffer should also be added to the wells on both sides of the Marker to ensure that the electrophoresis bands are aligned. Transfer: Prepare a PVDF membrane of appropriate size, activate it in methanol for 15 s, wash it with double-distilled water for 2 min, and place it in transfer buffer for later use; wet the filter paper and sponge pad with transfer buffer for later use. Assemble the "sandwich" transfer structure in the following order: negative electrode—sponge pad—filter paper—gel—membrane—filter paper—sponge pad—positive electrode. After assembling the transfer tank, transfer the membrane in an ice bath at 100 V for 2 h. Blocking: After the transfer, the pre-stained Marker imprint on the gel should disappear and be clearly visible on the PVDF membrane. Remove the PVDF membrane with tweezers, rinse it twice in TBST, and block it in 5% BSA solution at room temperature for 3 h. Primary antibody incubation: After blocking, wash the membrane three times with TBST for 10 min each time. Cut the PVDF membrane to the molecular weight of the protein to be detected, place it in an antibody incubation chamber, and incubate overnight at 4°C with a slow shaker. Secondary antibody incubation: Wash the membrane three times with TBST, 10 min each time. Incubate for 1.5 h at room temperature with HRP-labeled secondary antibody diluted 1:5000. Exposure: Wash the membrane three times with TBST, 10 min each time. Add freshly prepared ECL chemiluminescence reagent to the membrane, expose it using a chemiluminescence imaging system, record and save the results.

[0077] The results are as follows Figure 7 , Figure 8 As shown, the trispecific T cell binder can effectively reduce the phosphorylation of Akt protein in SK-BR-3 cells and NCI-N87 cells.

[0078] Example 3: Effect of CCK-8 assay on the proliferation capacity of target cells by the trispecific T cell conjugate

[0079] The effect of the trispecific T cell conjugate on the proliferation of target cells was analyzed using CCK-8 assay. SK-BR-3, NCI-N87, and MDA-MB-453 cells were used as target cells. After co-incubating the trispecific T cell conjugate with each target cell for 48 h, the effect of the antibody on the target cell viability was detected by CCK-8 assay.

[0080] Cells were digested and counted, then transferred to 96-well plates with 3000 cells per well. Cells were incubated overnight to allow adherence. The next day, the culture supernatant was aspirated, and 500 nM of a 2-fold serially diluted trispecific T-cell conjugate was added. Cells were incubated for 48–72 h. When the cell confluence in the control group (without the trispecific T-cell conjugate) reached 80%, the culture medium was aspirated, and the cells were washed twice with PBS. 100 μL of a 1:10 diluted CCK-8 reagent was added to each well, and the cells were incubated for 1–4 h. The absorbance at 450 nm was measured using a microplate reader. Three cell-free wells were reserved as blanks. Data processing: The antibody's cell growth inhibition efficiency was calculated using the following formula: Cell inhibition rate = (Control wells − Experimental wells) ÷ (Control wells − Blank wells) × 100%.

[0081] The results are as follows Figure 9 As shown, the trispecific T cell conjugate can significantly inhibit the proliferation of SK-BR-3, NCI-N87 and MDA-MB-453 cells.

[0082] Example 4: Immunofluorescence detection of the ability of trispecific T cell binders to mediate immune synapse formation

[0083] To investigate the binding ability of the trispecific T cell binder to tumor cells, the immune synapse formation ability was studied.

[0084] Coat a confocal dish with Poly-L-Lysine. Dilute the poly-L-Lysine solution to 0.1 mg / mL with sterile water, and spread an appropriate amount of solution to cover the bottom of the dish. Coat the confocal dish at room temperature for 1 h. 3) Aspirate the poly-L-Lysine solution and allow it to dry completely in a laminar flow hood. Rinse twice with PBS solution and air dry for later use. Take 2×10 5SK-BR-3 cells were resuspended in 200 μL of 100 nM START antibody and incubated in a 37°C cell culture incubator for 1 h. PBMC cells were resuscitated according to protocol, preheated to 37°C in a water bath, thawed, sterilized, and transferred to a clean bench. Cells were transferred to 15 mL centrifuge tubes containing 8 mL of preheated 1640 complete medium, and the tubes were rinsed 2-3 times with 2 mL of complete medium to minimize cell loss. The medium was then brought to a final volume of 14 mL. The cells were centrifuged at 400×g for 10 min at room temperature, and the supernatant was carefully discarded. The cells were gently resuspended in 1 mL of complete medium, and then brought to the desired volume. A suitable amount of cells was collected for counting and viability testing. The cells were centrifuged at 400×g for 5 min, the supernatant was discarded, and 1×10⁻⁶ cells were added to the centrifuge tube. 5 One PBMC cell line was mixed thoroughly with target cells and added to a confocal dish coated with poly-L-lysine. The cells were then incubated for 3 hours. The culture medium was discarded, and the cells were washed twice with PBS. 4% PFA solution was then added, and the cells were fixed at room temperature for 10 minutes. The 4% PFA solution was discarded, and the cells were washed three times with PBS for 2 minutes each time. An appropriate amount of PBS solution containing 0.1% Triton X-100 was added, and the cells were permeabilized at room temperature for 5 minutes. The supernatant was discarded, and the cells were washed three times with PBS for 2 minutes each time. 5% BSA was added, and the cells were blocked at room temperature for 1 hour, followed by two washes with PBS. CD3 antibody (OKT3) diluted 1:100 was added, and the cells were incubated overnight at 4°C. The next day, the cells were washed three times with PBS, and goat anti-mouse IgG-FITC and F-actin staining reagents were added. The cells were incubated at room temperature for 2 hours. The cells were washed three times with PBS, and an appropriate amount of PBS solution was added before imaging. The results are shown below. Figure 10 As shown, the trispecific T cell binder can mediate the formation of immune synapses between T cells and tumor cells.

[0085] Example 5: Flow cytometry detection of the ability of the trispecific T cell conjugate to activate T cells.

[0086] Using PBMCs as the target, we investigated the ability of the trispecific T cell binder to activate T cells.

[0087] Digest and count the cells, transfer them to 24-well plates, adding 20,000 cells per well, and incubate overnight to allow cell adhesion. Discard the culture supernatant and add different concentrations of trispecific T-cell conjugates. Ensure sufficient volume is reserved for PBMCs. Resuscitate PBMCs according to protocol. Add 1 × 10⁵ PBMCs per well at an E:T (effect cell:tumor cell) ratio of 5:1, and incubate for another 48 h. Discard the culture supernatant, repeatedly pipetting and repositioning the cells within the wells to remove as much PBMC residue as possible. Centrifuge at 600 × g for 10 min, wash cells with 200 μL of flow cytometry buffer, repeating twice. Resuspend the cells in 50 μL of Cell Staining Buffer containing flow cytometry antibodies and incubate at 4°C for 0.5 h. Wash twice, resuspend the cells in 300 μL of flow cytometry buffer, and perform flow cytometry analysis.

[0088] The results are as follows Figure 11 , 12 As shown, the trispecific T cell binder can mediate the enhancement of T cell activation markers CD69 and CD25, demonstrating that it can activate T cells in the presence of tumor cells.

[0089] Example 6: CCK-8 assay for T cell-dependent cytotoxicity mediated by the trispecific T cell conjugate.

[0090] The cytotoxic effect of the trispecific T cell conjugate on T cell-dependent cells was analyzed using CCK-8 assay, with SK-BR-3 and NCI-N87 cells as target cells. After co-incubating the trispecific T cell conjugate and PBMCs with each target cell, the effect on target cell viability was assessed using the CCK-8 assay.

[0091] The specific steps include the following: (1) Digest and count the cells, transfer them to a 96-well plate, add 5000 cells to each well, and incubate overnight in an incubator to allow the cells to adhere. (2) Discard the culture supernatant and add 2 μM of initial, four-fold serially diluted antibody. Note that the volume of PBMCs should be reserved. (3) Resuscitate PBMC cells according to the operating procedure. (4) Add 5 × 10⁶ cells to each well according to the E:T (effect cells: tumor cells) ratio of 10:1. 4(5) Discard the culture medium supernatant, wash twice with PBS, and try to wash away the residual PBMCs, but be careful not to wash away the tumor cells. (6) Add 100 μL of 1:10 diluted CCK-8 reagent to each well, put it back into the incubator and incubate for 1~4 h, and detect the absorbance at 450 nm under a microplate reader. Reserve three cell-free detection wells as blank values. (7) Process the data and calculate the cell viability according to the following formula: Cell viability = (experimental wells − blank wells) ÷ (control wells − blank wells) × 100%.

[0092] The results are as follows Figure 13 As shown, the trispecific T cell conjugate can significantly mediate the killing of SK-BR-3 cells and NCI-N87 cells by T cells.

[0093] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A trispecific T-cell conjugate targeting HER2, HER3, and CD3, characterized in that: It includes the HER2 antigen-binding domain, the HER3 antigen-binding domain, and the CD3 antigen-binding domain.

2. The trispecific T-cell conjugate targeting HER2, HER3, and CD3 according to claim 1, characterized in that: The HER2 antigen-binding domain, HER3 antigen-binding domain, and CD3 antigen-binding domain of bacteria include heavy chain variable regions and light chain variable regions. The heavy chain variable regions and light chain variable regions of the CD3 antigen-binding domain are cross-connected with the heavy chain variable regions and light chain variable regions of the HER2 / HER3 antigen-binding domain, and then connected upstream to the heavy chain variable regions and light chain variable regions of another HER3 / HER2 antigen-binding domain.

3. The trispecific T-cell conjugate targeting HER2, HER3, and CD3 according to claim 2, characterized in that: The HER2 antigen-binding domain comprises a heavy chain variable region HER2 VH and a light chain variable region HER2 VL; HER2 VH comprises one or more of the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID No. 20, SEQ ID No. 22, and SEQ ID No. 24, respectively; HER2 VL comprises one or more of the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID No. 14, SEQ ID No. 16, and SEQ ID No. 18, respectively. The HER3 antigen-binding domain comprises a heavy chain variable region HER3 VH and a light chain variable region HER3 VL; HER3 VH comprises one or more of the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID No. 32, SEQ ID No. 34, and SEQ ID No. 36, respectively; HER3 VL comprises one or more of the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID No. 26, SEQ ID No. 28, and SEQ ID No. 30, respectively. The CD3 antigen-binding domain comprises a heavy chain variable region CD3 VH and a light chain variable region CD3 VL; CD3 VH comprises one or more of the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID No. 44, SEQ ID No. 46, and SEQ ID No. 48, respectively; and CD3 VL comprises one or more of the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID No. 38, SEQ ID No. 40, and SEQ ID No. 42, respectively. Preferably, the HER2 antigen-binding domain includes a heavy chain variable region as shown in SEQ ID No. 3 and a light chain variable region as shown in SEQ ID No. 1; the HER3 antigen-binding domain includes a heavy chain variable region HER3 VH as shown in SEQ ID No. 7 and a light chain variable region HER3 VL as shown in SEQ ID No. 5; and the CD3 antigen-binding domain includes a heavy chain variable region CD3 VH as shown in SEQ ID No. 11 and a light chain variable region CD3 VL as shown in SEQ ID No.

9.

4. The trispecific T-cell conjugate targeting HER2, HER3, and CD3 according to claim 2, characterized in that: In the trispecific T-cell conjugate, the heavy chain variable region and the light and heavy chain variable regions of each antigen-binding domain are connected by linker peptides. The linker peptide sequences at different positions may be the same, partially the same, or different, and the linker peptide sequences are shown in any of SEQ ID No. 49-52.

5. The trispecific T-cell conjugate targeting HER2, HER3, and CD3 according to claim 4, characterized in that: The three-specific T-cell conjugate comprises, in sequence, a first functional region, a second functional region, a third functional region, a fourth functional region, a fifth functional region, and a sixth functional region; each functional region is connected by a linker peptide; The first and second functional areas each include either VH_HER3 or VL_HER3; the third, fourth, fifth, and sixth functional areas each include one of VL_HER2, VH_CD3, VH_HER2, and VL_CD3, wherein VL_HER2 and VH_HER2 are not directly connected, and VH_CD3 and VL_CD3 are not directly connected. Alternatively, the first and second functional areas may each include VH_HER2 or VL_HER2; the third, fourth, fifth, and sixth functional areas may each include one of VL_HER3, VH_CD3, VH_HER3, and VL_CD3, wherein VL_HER3 and VH_HER3 are not directly connected, and VH_CD3 and VL_CD3 are not directly connected.

6. The trispecific T-cell conjugate targeting HER2, HER3, and CD3 according to claim 5, characterized in that: The first functional region and the second functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 49; the second functional region and the third functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 50; the third functional region and the fourth functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 51; the fourth functional region and the fifth functional region are connected by a linker peptide with the sequence shown in SEQ ID No. 52; and the fifth functional region and the sixth functional region are connected by a linker peptide with the sequence shown in SEQ ID No.

51.

7. The method for preparing the trispecific T-cell conjugate targeting HER2, HER3, and CD3 as described in any one of claims 1-6, characterized in that: An expression vector capable of expressing the three-specific T cell conjugate was constructed, and the expression vector was transfected into host cells. After culturing, the expression product was isolated and purified to obtain a protein solution containing the three-specific T cell conjugate.

8. The preparation method according to claim 7, characterized in that: When constructing the expression vector, histidine tags and Strep tag-II tags were added to the N-terminus and C-terminus of the gene fragment expressing the trispecific T cell binder, respectively; the expression vector was pET28a(−), and the host cell was the Escherichia coli prokaryotic expression system.

9. The use of the trispecific T-cell binder targeting HER2, HER3 and CD3 as described in any one of claims 1-6 in a drug for treating tumors or related diseases caused by HER2-specific antigen expression.

10. The application according to claim 9, characterized in that: The tumor may be breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, and soft tissue cancer; preferably, the tumor is selected from breast cancer and gastric cancer.

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