CN116239700B - Tumor dual-targeting trispecific T cell adapter and application thereof - Google Patents

Abstract

The present invention discloses the preparation and application of a tumor dual-targeting tri-specific T cell adapter, and relates to the field of biopharmaceutical technology. The tumor dual-targeting tri-specific T cell adapter of the present invention has better specific T cell activation, proliferation activity and T cell anti-tumor activity at the cellular level, and can be more effectively aggregated inside solid tumors, which is beneficial to the treatment of solid tumors. Therefore, the tri-specific T cell adapter of the present invention or the gene encoded by it can be used to prepare anti-tumor drugs. The preparation method of the tumor dual-targeting tri-specific T cell adapter has the advantages of simple preparation and high expression level.

Description

A tumor dual-targeting trispecific T cell engager and its application

Technical Field

The present invention relates to the field of biopharmaceutical technology, and in particular to the preparation and application of a tumor dual-targeting tri-specific T cell adapter.

Background technique

Cancer has become a major disease that endangers human health. In recent years, T cell engagers have become one of the most popular antibody anti-tumor drugs, showing good anti-tumor effects. Its mechanism of action is mainly through targeting tumor antigens and T cell CD3 domains to form a "tumor cell-T cell engager-T cell" immune synapse, mediating T cell participation in inducing downstream signal transduction events, activating T cell cytotoxic mechanisms (including cytokine release, etc.), and leading to tumor cell death.

Target selection is one of the key factors in the success of T cell engager therapy for tumors. Currently, the targets of T cell engagers are mainly limited to the extracellular region of membrane proteins. Not only is the selection range very limited (membrane proteins account for ~8% of total cell proteins), but the specificity is not strong. Therefore, the exploration of new targets has become one of the important tasks in the development of T cell engagers. Intracellular proteins (including tumor mutant proteins) can be degraded into antigenic peptides in the proteasome, and then presented to the cell surface by HLA class I molecules to form a peptide-HLA complex (pHLA), which is recognized by the T cell receptor (TCR).

Currently, TCR therapy can target intracellular and extracellular antigens, but this antigen peptide fragment must be presented on tumor cells by binding to a specific pHLA complex. Therefore, the primary goal of the research is to identify pHLA complexes that are highly expressed in tumors but rare in healthy tissues and have high TCR affinity.

Since pHLA has a wide range of target selection and strong specificity, and TCR has high specificity in pHLA recognition due to thymic selection, TCR-based T cell engagers are highly promising tumor immunotherapy drugs. However, the expression level of pHLA is extremely low, and a single cell presents a few to several thousand specific pHLAs, which is much lower than traditional tumor surface highly expressed antigens (tens of thousands to hundreds of thousands), which greatly affects the in vivo targeting and anti-tumor activity of TCR-based T cell engagers. The trispecific T cell engager targeting pHLA and traditional tumor surface highly expressed antigens has the characteristics of both pHLA and traditional tumor surface highly expressed antigens, and is one of the good strategies to solve the low expression of pHLA. However, this method has not been reported yet, and there are many problems involved that need to be solved urgently, so a TCR-based tumor dual-targeting T cell engager with good specificity needs to be developed.

Summary of the invention

The present invention provides a preparation method and application of a TCR-based tri-specific T cell adapter with TCR and EGFR as target antigens.

The specific technical scheme of the present invention is as follows:

The present invention provides a tumor dual-targeting trispecific T cell engager, comprising a framework comprising an IgG antibody constant region, wherein the framework comprises two heavy chains and two light chains, wherein the heavy chain comprises heavy chain constant regions CH1, CH2, and CH3, and the light chain comprises CL, and the tumor dual-targeting trispecific T cell engager is a heterotetramer, which is in any of the following forms:

(1) The first subunit includes the variable region, constant region and one light chain of the T cell receptor α chain, the second subunit includes the variable region, constant region and one heavy chain of the T cell receptor β chain, the third subunit includes the heavy chain variable region of the anti-EGFR antibody and another heavy chain of the framework, and the fourth subunit includes the light chain variable region of the anti-EGFR antibody and another light chain of the framework; wherein the heavy chain variable region of the anti-EGFR antibody in the third subunit and the light chain variable region of the anti-EGFR antibody in the fourth subunit constitute the anti-EGFR antibody domain; the first subunit or the fourth subunit is also connected to the heavy chain variable region and the light chain variable region of the anti-CD3 antibody;

(2) the first subunit comprises the variable region and constant region of the α chain of the T cell receptor and a light chain in the framework, the second subunit comprises the variable region and constant region of the β chain of the T cell receptor and a heavy chain in the framework, the third subunit comprises the heavy chain variable region of the anti-CD3 antibody and another heavy chain in the framework, and the fourth subunit comprises the light chain variable region of the anti-CD3 antibody and another light chain in the framework; wherein the heavy chain variable region of the anti-CD3 antibody in the third subunit and the light chain variable region of the anti-CD3 antibody in the fourth subunit constitute the anti-CD3 antibody domain; the two heavy chains in the framework are also connected to the heavy chain variable region and light chain variable region of the anti-EGFR antibody;

In the above (1) and (2), the variable region and constant region of the T cell receptor α chain in the first subunit and the variable region and constant region of the T cell receptor β chain in the second subunit constitute a T cell receptor domain for recognizing the antigen peptide-HLA complex. The IgG antibody is a human IgG antibody. The heavy chain includes constant regions CH1, CH2, and CH3, and the constant region also includes a hinge region.

The first subunit and the second subunit of the trispecific T cell engager of the present invention contain a T cell receptor for recognizing an antigen peptide-HLA complex, wherein the antigen peptide is formed by degradation of a tumor mutant protein in the proteasome, and also contains an anti-EGFR antibody. The trispecific T cell engager targeting pHLA and traditional tumor surface highly expressed antigens further targets the T cell CD3 domain to tumor antigens through an anti-CD3 antibody, mediates T cell participation in inducing downstream signal transduction events, and activates the cytotoxic mechanism of T cells (including cytokine release, etc.). The results of the embodiments of the present invention show that the tumor dual-targeting trispecific T cell engager has better specific T cell activation, proliferation activity and T cell anti-tumor activity at the cellular level, and can effectively cause tumor cell death.

As an example, the polypeptide sequence loaded by the polypeptide-HLA complex is SLLMWITQC; in the embodiment of the present invention, as an example, the anti-EGFR antibody is selected as cetuximab antibody, and when used, any anti-EGFR antibody is acceptable, and the anti-CD3 antibody is anti-CD3e antibody. But it is not limited thereto.

Preferably, the two CH3s in the second subunit and the third subunit are designed into a knob structure and a hole structure respectively using the knob-into-hole technology. Among them, the CH3 domains of the second subunit and the third subunit introduce LALA-PG mutations to silence the antibody Fc fragment, lose the function of binding to the Fc receptor, and avoid the toxic side effects of nonspecific killing caused by Fc.

Specifically, when the trispecific T cell engager is (1) and the heavy chain variable region and light chain variable region of an anti-CD3 antibody are connected to the first subunit, the amino acid sequence of the first subunit is shown in SEQ ID No.20, the amino acid sequence of the second subunit is shown in SEQ ID No.21, the amino acid sequence of the third subunit is shown in SEQ ID No.22, and the amino acid sequence of the fourth subunit is shown in SEQ ID No.23;

When the trispecific T cell engager is (2) and a heavy chain in the skeleton of the second subunit is connected to the heavy chain variable region and the light chain variable region of the anti-EGFR antibody, the amino acid sequence of the first subunit is shown in SEQ ID No.15, the amino acid sequence of the second subunit is shown in SEQ ID No.16, the amino acid sequence of the third subunit is shown in SEQ ID No.17, and the amino acid sequence of the fourth subunit is shown in SEQ ID No.18.

The present invention also provides a gene encoding the trispecific T cell engager. When the trispecific T cell engager is (1) and the first subunit is connected to the heavy chain variable region and the light chain variable region of the anti-CD3 antibody, the nucleotide sequence of the first subunit is shown as SEQ ID No.6, the nucleotide sequence of the second subunit is shown as SEQ ID No.7, the nucleotide sequence of the third subunit is shown as SEQ ID No.8, and the nucleotide sequence of the fourth subunit is shown as SEQ ID No.9; when the trispecific T cell engager is (2) and a heavy chain in the skeleton of the second subunit is connected to the heavy chain variable region and the light chain variable region of the anti-EGFR antibody, the nucleotide sequence of the first subunit is shown as SEQ ID No.1, the nucleotide sequence of the second subunit is shown as SEQ ID No.2, the nucleotide sequence of the third subunit is shown as SEQ ID No.3, and the nucleotide sequence of the fourth subunit is shown as SEQ ID No.4.

The present invention also provides a method for preparing the trispecific T cell adapter, comprising the following steps: (1) constructing a gene vector expressing the coding gene of the first subunit, the coding gene of the second subunit, the coding gene of the third subunit and the coding gene of the fourth subunit; (2) transfecting the gene vector expressing the coding gene of the first subunit, the coding gene of the second subunit, the coding gene of the third subunit and the coding gene of the fourth subunit in step (1) into mammalian cells, culturing and purifying the protein to obtain the trispecific T cell adapter. Preferably, the vector in step (1) is pLVX-Puro; the mammalian cell in step (2) is a 293F cell. The present invention also provides the use of the trispecific T cell adapter in the preparation of an anti-tumor drug. The present invention also provides the use of the gene in the preparation of an anti-tumor drug. The present invention also provides an anti-tumor drug, the active ingredient of which is the trispecific T cell adapter or the gene.

Beneficial effects of the present invention: The tumor dual-targeting trispecific T cell engager of the present invention has better specific T cell activation, proliferation activity and T cell anti-tumor activity at the cellular level, and can more effectively aggregate inside solid tumors, which is beneficial to the treatment of solid tumors. Therefore, the trispecific T cell engager of the present invention or the gene encoded by it can be used to prepare anti-tumor drugs. The preparation method of the tumor dual-targeting trispecific T cell engager has the advantages of simple preparation and high expression level.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a molecular diagram of a tumor dual-targeting trispecific T cell engager and its control molecule.

Figure 2 is a deglycosylation analysis diagram of the tumor dual-targeting trispecific T cell engager and its control molecule. Among them, (a) deglycosylation analysis diagram of tNY-aEGFR/aCD3 and tNY/aCD3, Lane 1: tNY/aCD3; Lane 2: tNY/aCD3 treated with PNGase; Lane 3: tNY-aEGFR/aCD3; Lane 4: tNY-aEGFR/aCD3 treated with PNGase; (b) deglycosylation analysis diagram of tNY-aCD3/aEGFR and control molecules; Lane 1: tNY-aCD3/aEGFR treated with PNGase; Lane 2: tNY-aCD3/aEGFR; Lane 3: tCrtl-aCD3/aEGFR treated with PNGase; Lane 4: tCrtl-aCD3/aEGFR; Lane 5: tNY-aCD3/aCtrl treated with PNGase; Lane 6: tNY-aCD3/aCtrl; Lane 7: PNGase; Lane 8: tNY/aEGFR; Lane 9: tNY/aEGFR treated with PNGase.

Figure 3 shows the specific affinity analysis of the tumor dual-targeting trispecific T cell engager and its control molecule to NY-ESO-1SLL/HLA-A*02:01. (a) Affinity analysis of tNY-aEGFR/aCD3 and tNY/aCD3 to NY-ESO-1 SLL/HLA-A*02:01; (b) Affinity analysis of tNY-aEGFR/aCD3 and tNY/aCD3 to NY-ESO-1 SLL/HLA-A*02:01; (c) Affinity analysis of tNY-aCD3/aEGFR and its control molecule to NY-ESO-1 SLL/HLA-A*02:01; (d) Affinity analysis of tNY-aCD3/aEGFR and its control molecule to NY-ESO-1 SLL/HLA-A*02:01.

Figure 4 shows the affinity analysis of the tumor dual-targeting trispecific T cell engager to PC-9 cells (EGFR ⁺ ). (a) Affinity analysis of tNY-aEGFR/aCD3 and tNY/aCD3 to PC-9 cells; (b) Affinity analysis of tNY-aCD3/aEGFR and its control molecules to PC-9 cells.

Figure 5 shows the affinity analysis of the tumor dual-targeting trispecific T cell engager to Jurkat cells (CD3 ⁺ ). (a) Affinity analysis of tNY-aEGFR/aCD3 and tNY/aCD3 to Jurkat cells; (b) Affinity analysis of tNY-aCD3/aEGFR and its control molecules to Jurkat cells.

Figure 6 shows that the tumor dual-targeted trispecific T cell engager mediates PBMC killing of A375-NY cells. (a) tNY-aEGFR/aCD3 and tNY/aCD3 mediate PBMC killing of A375-NY cells; (b) tNY-aCD3/aEGFR and its control molecules mediate PBMC killing of A375-NY cells.

Figure 7 shows the specific activation of T cells stimulated by tNY-aCD3/aEGFR. (a) The proportion of T cells activated in the early stage; (b) The proportion of T cells activated in the late stage.

FIG. 8 shows tNY-aCD3/aEGFR stimulating T cell expansion.

FIG. 9 shows that tNY-aCD3/aEGFR stimulates T cells to release interferon-γ.

Figure 10 shows the specific anti-tumor activity of tNY-aCD3/aEGFR in vivo, where * represents p<0.05, and ** represents p<0.01.

Detailed ways

The animal experiments in this application were approved by the Experimental Animal Welfare Ethics Review Committee of Zhejiang University, application number 12435. The human experiments in this application were approved by the Medical Ethics Committee of the School of Pharmacy of Zhejiang University, approval number: (2018) Lunyanpi No. 003.

Example 1

Preparation of trispecific T cell engagers for dual tumor targeting.

TCR selected NY-TCR (TCR targeting SLLMWITQC polypeptide and HLA-A*0201 complex, referred to as tNY), from patent: US20110014169A1; anti-EGFR antibody selected cetuximab (referred to as aEGFR, light chain variable region nucleotide sequence from NCBI, sequence number: KX138561, heavy chain variable region nucleotide sequence from NCBI, sequence number: KX138562.1); anti-CD3 antibody selected HXR32, from patent: WO2012162067A2. A total of two different molecular forms of tumor dual-targeting trispecific T cell engagers were designed (Figure 1a and c, where b is the control T cell engager of a, d, e, and f are the control T cell engagers of c), and 14 human renal epithelial cell (293F) expression plasmids were constructed.

tNY-aEGFR/aCD3: tNYECDα-GS-CL-His ₆ (gene sequence as shown in SEQ ID No.29, SEQ ID No.1 and SEQ ID No.32), tNYECDβ-GS-CH1-CH2-CH3(knob)-(G4S) ₃ -aEGFRVH-(G4S) ₃ -aEGFRVL (gene sequence as shown in SEQ ID No.29 and SEQ ID No.2), aCD3VH-CL-CH2-CH3(hole) (gene sequence as shown in SEQ ID No.29 and SEQ ID No.3), aCD3VL-CH1-Flag (gene sequence as shown in SEQ ID No.29, SEQ ID No.4 and SEQ ID No.34);

tNY/aCD3: tNYECDα-GS-CL-His ₆ (gene sequence as shown in SEQ ID No.29, SEQ ID No.1 and SEQ ID No.32), tNYECDβ-GS-CH1-CH2-CH3 (knob) (gene sequence as shown in SEQ ID No.29 and SEQ ID No.5), aCD3VH-CL-CH2-CH3 (hole) (gene sequence as shown in SEQ ID No.29 and SEQ ID No.3), aCD3VL-CH1-Flag (gene sequence as shown in SEQ ID No.29, SEQ ID No.4 and SEQ ID No.34);

tNY-aCD3/aEGFR: tNYECDα-GS-LC-(G4S) ₃ -aCD3VH-aCD3VL (gene sequence as shown in SEQ ID No.29 and SEQ ID No.6), tNYECDβ-GS-CH1-CH2-CH3(knob)-His ₆ (gene sequence as shown in SEQ ID No.29, SEQ ID No.7 and SEQ ID No.32), aEGFRVH-CH1-CH2-CH3(hole)-Flag (gene sequence as shown in SEQ ID No.29, SEQ ID No.8 and SEQ ID No.34), aEGFRVL-CL (gene sequence as shown in SEQ ID No.29 and SEQ ID No.9).

tCrtl-aCD3/aEGFR: tCtrlECDα-GS-LC-(G4S)3-aCD3VH-aCD3VL (gene sequence as shown in SEQ ID No.30 and SEQ ID No.10), tCtrlECDβ-GS-CH1-CH2-CH3(knob)-His ₆ (gene sequence as shown in SEQ ID No.31, SEQ ID No.11 and SEQ ID No.32), aEGFRVH-CH1-CH2-CH3(hole)-Flag (gene sequence as shown in SEQ ID No.29, SEQ ID No.8 and SEQ ID No.34), aEGFRVL-CL (gene sequence as shown in SEQ ID No.29 and SEQ ID No.9).

tNY-aCD3/aCtrl: tNYECDa-GS-LC-(G4S) ₃ -aCD3VH-aCD3VL (gene sequence as shown in SEQ ID No.29 and SEQ ID No.6), tNYECDβ-GS-CHl-CH2-CH3(knob)-His ₆ (gene sequence as shown in SEQ ID No.29, SEQ ID No.7 and SEQ ID No.32), aCtrlVH-CH1-CH2-CH3(hole)-Flag (gene sequence as shown in SEQ ID No.29, SEQ ID No.12 and SEQ ID No.34), aCtrlVL-CL (gene sequence as shown in SEQ ID No.29 and SEQ ID No.13).

tNY/aEGFR: tNYECDα-GS-LC (gene sequence as shown in SEQ ID No.29 and SEQ ID No.14), tNYECDβ-GS-CH1-CH2-CH3 (knob)-His ₆ (gene sequence as shown in SEQ ID No.29, SEQ ID No.7 and SEQ ID No.32), aEGFRVH-CH1-CH2-CH3 (hole)-Flag (gene sequence as shown in SEQ ID No.29, SEQ ID No.8 and SEQ ID No.34), aEGFRVL-CL (gene sequence as shown in SEQ ID No.29 and SEQ ID No.9).

Among them, tNYECDα and tNYECDβ are the extracellular domains of the α chain and β chain of tNY, and tCtrlECDα and tCtrlECDβ are the extracellular domains of the α chain and β chain of tCtrl, respectively. (G4S) ₃ is a flexible linker with three GGGGS lengths, aEGFRVH and aEGFRVL are the heavy chain variable region and light chain variable region of anti-EGFR, aCtrlVH and aCtrlVL are the heavy chain variable region and light chain variable region of control antibody, aCD3VH and aCD3VL are the heavy chain variable region and light chain variable region of anti-CD3ε antibody, His ₆ is a tag composed of 6 histidines for protein purification, Flag tag is a tag composed of 8 amino acids DYKDDDDK for protein purification, CL, CH1, CH2, and CH3 are all IgG1 structures, and the knob and hole structures are designed between the two CH3s using knob-into-hole technology, respectively. At the same time, LALA-PG mutation is introduced into CH2 to silence Fc, and CD3VH-CL-CH2-CH3 (hole) subunit and CD3VL-CH1-Flag subunit are crossmab structures.

The α and β chain extracellular regions of tNY and tCtrl, the heavy chain variable regions and light chain variable regions of aEGFR, aCD3 and aCtrl, the heavy chain constant region of IgG1 antibody and the K light chain constant region were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. Through PCR and overlapping PCR, each gene segment was combined according to the requirements of the recombinant plasmid, and the Xho I restriction site (CTCGAG) and kozaka sequence (ACCACC) were introduced upstream of the recombinant gene, and the stop codon (TGA) and EcoRI restriction site (GAATTC) were introduced downstream, and ligated to the pLVX-Puro plasmid (the plasmid was also double-digested with XhoI and EcoRI) by T4 ligase. Afterwards, the recombinant plasmid was transiently transformed into 293F cells and stained with HisTrap HP affinity column (purchased from GE, catalog number: 17-5248-02) and anti- M1 affinity gel (purchased from Sigma, catalog number: A4596) was used for column chromatography purification to obtain the corresponding protein. The expression level of tNY-aCD3/aEGFR (about 3 mg/L) was much higher than that of tNY-aEGFR/aCD3 (expression level was about 300 μg/L). The purified product was deglycosylated with PNGase (purchased from Sigma, catalog number: PP0201) and analyzed by SDS-PAGE (the purified product without PNGase treatment was used as a control).

As shown in Figure 2, each subunit of the T cell engager treated with PNGase appeared at the theoretical size position (the theoretical molecular weight of each subunit of the T cell engager is shown in Table 1), among which the β chain and α chain of TCR changed their positions before and after PNGase treatment, indicating obvious glycosylation modification, with the α chain changing more and the glycosylation modification being more complex.

Table 1 Theoretical molecular weight of each subunit of the trispecific T cell engager and its control molecule for dual tumor targeting

Example 2

(1) Affinity verification

Specific affinity verification for NY-ESO-1 SLL/HLA-A*02:01 (ELISA).

First, biotinylated NY-ESO-1 SLL/HLA-A*02:01 complex (the complex of SLLMWITQC and HLA-A*0201, called NY-ESO-1/A02-Biotin), WT1RMF/HLA-A*02:01 complex (the complex of RMFPNAPYL and HLA-A*0201, called WT 1/A02-Biotin) and Tax LLF/HLA-A*02:01 complex (the complex of LLFGYPRYV and HLA-A*0201, called Tax/A02-Biotin) were prepared by renaturation method.

100 μL, 1 μg/mL streptavidin (purchased from Shanghai Shenggong Biotechnology Co., Ltd., catalog number: A100497) was coated overnight at 4°C in a 96-well EIA/RIA plate (purchased from Coming, catalog number: 03619013). After washing 4 times with PBST (PBS solution containing 0.05% Tween 20), it was blocked at 37°C for 1 hour (blocking solution: 5% skim milk powder in PBST solution). After washing 4 times with PBST, 100 μL, 1 μg/mL NY-ESO-1/A02-Biotin, WTl/A02-Biotin or Tax/A02-Biotin was incubated at 37°C for 1 hour. After washing 4 times with PBST, 100 μL of concentration gradient tumor dual-targeting trispecific T cell engagers and control molecules were incubated at 37°C for 1 hour. After washing with PBST for 4 times, the cells were incubated with horseradish peroxidase-labeled goat anti-human IgG (H+L) (purchased from Bio-Technology Co., Ltd., catalog number: A0201) for 1 h at 37°C. After washing with PBST for 5 times, 100 μL of TMB liquid substrate (purchased from Sigma, catalog number: T4444) was added to develop color for about 10 min, 100 μL of 2 M sulfuric acid was added to terminate the reaction, and the OD450 value was measured.

The results showed that tNY-aEGFR/aCD3, tNY/aCD3, tNY-aCD3/aEGFR, tNY-aCD3/aCtrl and tNY/aEGFR had high affinity for NY-ESO-1 SLL/HLA-A*02:01 due to the presence of tNYECD subunit, and were concentration-dependent; while tCrtl-aCD3/aEGFR had no affinity for NY-ESO-1 SLL/HLA-A*02:01 and could be used as a control molecule in subsequent drug evaluation (Figures 3a, 3c). tNY-aEGFR/aCD3, tNY/aCD3, tNY-aCD3/aEGFR, tNY-aCD3/aCtrl and tNY/aEGFR had no affinity for WT 1RMF/HLA-A*02:01 and TaxLLF/HLA-A*02:01, indicating that their affinity specificity for NY-ESO-1SLL/HLA-A*02:01 was good ( Figures 3b, 3d ).

(2) Affinity analysis for EGFR antigen

PC-9 cells (EGFR+ cells) in the logarithmic growth phase were collected by centrifugation at 500×g for 5 minutes, and 200 μL of different concentrations of tumor dual-targeting trispecific T cell engagers and their control molecules were incubated at 4°C for 30 minutes (with incubation in PBS as a control). After washing with PBS twice, 200 μL of FITC-labeled goat anti-human IgG (H+L) (purchased from Biyuntian Biotechnology Co., Ltd., catalog number: A0556) was incubated at 4°C. After washing with PBS twice, the cells were resuspended with 400 μL of PBS and detected using an ACEA NovoCyteTM flow cytometer.

The results are shown in Figure 4a. The mean fluorescence intensity of PC-9 cells incubated with tNY-aEGFR/aCD3 increases with the concentration, indicating that tNY-aEGFR/aCD3 can recognize EGFR antigen and is concentration-dependent; tNY/aCD3 does not contain aEGFR subunits and cannot recognize EGFR antigens, so it has no affinity for PC-9 cells. The mean fluorescence intensity of PC-9 cells incubated with tNY-aCD3/aEGFR, tCrtl-aCD3/aEGFR and tNY/aEGFR is significantly higher than that of tNY-aCD3/aCtrl and is concentration-dependent, indicating that tNY-aCD3/aEGFR, tCrtl-aCD3/aEGFR and tNY/aEGFR can recognize EGFR antigens on the surface of PC-9 cells; tNY-aCD3/aCtrl does not contain aEGFR subunits, so it has no affinity for PC-9 cells (Figure 4b).

(3) Affinity analysis for CD3 antigen

Jurkat cells (CD3 ⁺ cells) in the logarithmic growth phase were collected by centrifugation at 500×g for 5 minutes, and 200 μL of different concentrations of tumor dual-targeting trispecific T cell engagers and their control molecules were incubated at 4°C for 30 minutes (with incubation in PBS as a control). After washing with PBS twice, 200 μL of FITC-labeled goat anti-human IgG (H+L) (purchased from Bio-Technology Co., Ltd., catalog number: A0556) was incubated at 4°C. After washing with PBS twice, the cells were resuspended with 400 μL of PBS and detected using an ACEA NovoCyteTM flow cytometer.

The results showed that the affinity of tNY-aEGFR/aCD3 and tNY/aCD3 for Jurkat cells was consistent and concentration-dependent (Figure 5a). As shown in Figure 5b, the average fluorescence intensity of Jurkat cells incubated with tNY-aCD3/aEGFR, tNY-aCD3/aCtrl and tCrtl-aCD3/aEGFR was significantly higher than that of tNY/aEGFR, and was concentration-dependent, indicating that tNY-aCD3/aEGFR, tNY-aCD3/aCtrl and tCrtl-aCD3/aEGFR can recognize the CD3 antigen on the surface of Jurkat cells, but their affinity for CD3 is much weaker than that of tNY-aEGFR/aCD3. tNY/aEGFR does not contain aCD3 subunit, so it has no affinity for Jurkat cells.

Example 3: In vitro specific anti-tumor activity

A375-NY cells (NY-ESO-1SLL/HLA-A*02:01 ⁺ and EGFR ⁺ ) stably transfected with EGFP and presenting peptide SLLMWITQC were plated with PBMC (purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., catalog number: A10S045018) at a ratio of 1:4 in a 24-well cell culture plate, and a series of gradient concentrations of tNY/aCD3/aEGFR or tNY/aCD3 (n=3) were added, mixed and incubated in a 37°C, 5% CO ₂ incubator. After 48 hours, the cells were collected and stained with Annexin V, 633 apoptosis detection kit (purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., catalog number: AD11), and then the apoptosis of tumor cells was detected using ACEA NovoCyteTM flow cytometer. The results are shown in Figure 6a. Although tNY-aEGFR/aCD3 can mediate PBMC killing of A375-NY cells, the killing activity is not much different from tNY/aCD3, indicating that the molecular form of tNY-aEGFR/aCD3 is not the form that mediates T cells to produce the best anti-tumor activity.

A375-NY cells were plated with PBMCs at a ratio of 1:4 in 96-well cell culture plates, and a series of gradient concentrations of tNY-aCD3/aEGFR and its control molecules (n=3) were added, mixed and incubated in a 37°C, 5% CO ₂ incubator. After 48 hours, the culture supernatant was collected and detected with a lactate dehydrogenase detection kit (purchased from Bio-Technology Co., Ltd., catalog number: C0O17), and the EC50 was calculated using GraphPad software. As shown in Figure 6b, tNY-aCD3/aEGFR showed good PBMC-mediated killing activity against NY-ESO-1 SLL/HLA-A*02:01 and EGFR double-positive A375-NY cells, and the killing activity was concentration-dependent, and was much higher than tNY-aCD3/aCtrl. The fact that tCrtl-aCD3/aEGFR and tNY/aEGFR mediated only weak cytotoxic activity of PBMCs at high concentrations indicated that tNY-aCD3/aEGFR had good specificity.

Example 4: T cell specific activation detection

A375-NY cells and PBMC were plated in 48-well cell culture plates at a ratio of 1:4, and a series of gradient concentrations of tNY-aCD3/aEGFR and its control molecules (n=3) were added, mixed and incubated in a 37°C, 5% _CO2 incubator.

After 72 hours of culture, the cells were collected by centrifugation at 600×g for 5 minutes, washed once with 1×PBS, and resuspended in 1×PBS. 5μL APC-labeled mouse anti-human CD3 antibody (purchased from ThermoFisher Scientific, catalog number 17-0037-42), 5μL Super Bright 436-labeled mouse anti-human CD69 antibody (purchased from ThermoFisherScientific, catalog number 62-0699-42) and 1μL PE-labeled mouse anti-human CD25 antibody (purchased from ThermoFisherScientific, catalog number 12-0259-80) were added, and incubated at 4°C in the dark for 30 minutes. After washing twice with 1×PBS, the cells were resuspended in 200μL 1×PBS, and the proportion of CD69 and CD25 positive T cells (CD3 positive) was detected using CytomicFC 500MCL flow cytometer.

The results are shown in Figure 7. CD69 and CD25 are the main markers of early and late activation of T cells, respectively. Co-incubation of tNY-aCD3/aEGFR with A375-NY can specifically and significantly activate T cells, and the activation ratio is much higher than that of various control molecules. Even at an extremely low concentration of 20pM, co-incubation of tNY-aCD3/aEGFR with A375-NY can significantly activate T cells.

Example 5: T cell proliferation detection

PBMCs were collected by centrifugation at 600×g for 10 min, washed twice with 1×PBS preheated at room temperature, resuspended in 1×PBS, and CFSE (purchased from ThermoFisher Scientific, catalog number: 65-0850-84) was added to a final concentration of 2.5 μM, mixed, and incubated at 37°C in the dark for 15 min. A375-NY and CFSE-treated PBMCs were collected by centrifugation at 600×g for 10 min, washed once with PBS, and A375-NY and CFSE-treated PBMCs were plated in 48-well cell culture plates at a ratio of 1:4, and a series of gradient concentrations of tNY-aCD3/aEGFR and its control molecules (n=3) were added, mixed, and incubated in a 37°C, 5% _CO2 incubator.

After 96 h of culture, the cells were collected by centrifugation at 600 × g for 5 min, washed twice with 1 × PBS, and resuspended in 100 μL 1 × PBS. 5 μL PI stock solution (1 mg/mL) was added. After incubation at room temperature in the dark for 15 min, the cells were centrifuged at 600 × g for 5 min, resuspended in 400 μL 1 × PBS, and PBMCs were selected based on cell morphology using ACEA NovoCyteTM flow cytometer to detect their proliferation.

After activation, T cells can divide and proliferate. CFSE can non-specifically bind to intracellular proteins, produce green fluorescence, and distribute it evenly to two daughter cells as the cells divide. The green fluorescence decreases step by step as the number of divisions increases. The results are shown in Figure 8. Compared with the blank control group, tNY-aCD3/aEGFR co-incubated with A375-NY can stimulate the proliferation of T cells, and the proliferation ratio is proportional to the administration concentration; compared with tNY-aCD3/aEGFR, tNY-aCD3/aCtrl, tCrtl-aCD3/aEGFR and tNY/aEGFR can only stimulate T cell proliferation at a high concentration of 2000pM when co-incubated with A375-NY, and the proliferation multiple is much lower than tNY-aCD3/aEGFR, indicating that tNY-aCD3/aEGFR co-incubated with A375-NY has a strong ability to stimulate the proliferation of T cells and good specificity.

Example 6: Detection of interferon-γ

A375-NY cells and PBMC were plated in 48-well cell culture plates at a ratio of 1:4, and a series of gradient concentrations of tNY-aCD3/aEGFR and its control molecules (n=3) were added, mixed and incubated in a 37°C, 5% _CO2 incubator.

After 72 hours of culture, the supernatant was collected by centrifugation at 600×g for 5 minutes, diluted to a certain concentration, and the release of T cell interferon-γ was detected using an interferon-γ detection kit (purchased from ThermoFisher Scientific, catalog number: 88-7316-88).

The results are shown in Figure 9. Interferon-γ is a cytokine released after T cells are activated and has a strong tumor-suppressing effect. After co-incubation with A375-NY cells, tNY-aCD3/aEGFR can significantly stimulate T cells to release a large amount of interferon-γ, and it is concentration-dependent; although tNY-aCD3/aCtrl and A375-NY cells can also stimulate T cells to release interferon-γ, the release amount is significantly lower than tNY-aCD3/aEGFR; tCrtl-aCD3/aEGFR and A375-NY cells can basically not stimulate T cells to release interferon-γ after co-incubation, indicating that tNY-aCD3/aEGFR and tNY-aCD3/aCtrl have good specificity.

Example 7: In vivo specific anti-tumor activity of tNY-aCD3/aEGFR

A375 cells and PBMC were inoculated subcutaneously in the right axilla of 8-week-old female SCID-Beige mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., SPF grade) at a ratio of 2×10 ⁶ :2×10 ^6. One hour after inoculation, the mice were randomly divided into 4 groups (normal saline group, tCrtl-aCD3/aEGFR group, tNY-aCD3/aCtrl group, and tNY-aCD3/aEGFR group), with 4 mice in each group, and administered via the tail vein (100 μg/kg, the normal saline group was given an equal volume of PBS). After that, the drug was administered once every 3 days, for a total of 3 times. The size of the mouse tumor was measured and recorded (tumor volume = tumor length × tumor width × tumor width/2).

As shown in Figure 10, the tumor volume of mice in the tNY-aCD3/aEGFR group was significantly smaller than that in the saline group, tCrtl-aCD3/aEGFR group and tNY-aCD3/aCtrl group, indicating that tNY-aCD3/aEGFR can effectively inhibit the growth of mouse transplanted tumors at a lower dose with good specificity; compared with the saline group and tCrtl-aCD3/aEGFR group, the tNY-aCD3/aCtrl group had a certain effect of inhibiting tumor growth, but the in vivo anti-tumor effect was significantly inferior to that of the tNY-aCD3/aEGFR group.

Claims (5)

1. A tumor dual-targeting trispecific T cell engager, comprising a framework comprising an IgG antibody constant region, wherein the framework comprises two heavy chains and two light chains, wherein the heavy chain comprises heavy chain constant regions CH1, CH2, and CH3, and the light chain comprises CL, wherein the tumor dual-targeting trispecific T cell engager is a heterotetramer, The first subunit is the variable region, constant region and a light chain of the T cell receptor α chain, the second subunit is the variable region, constant region and a heavy chain of the T cell receptor β chain, the third subunit is the heavy chain variable region of the anti-EGFR antibody and another heavy chain in the framework, and the fourth subunit is the light chain variable region of the anti-EGFR antibody and another light chain in the framework; the heavy chain variable region of the anti-EGFR antibody in the third subunit and the light chain variable region of the anti-EGFR antibody in the fourth subunit constitute the anti-EGFR antibody domain; the first subunit is also connected to the heavy chain variable region and light chain variable region of the anti-CD3 antibody; The variable region and constant region of the T cell receptor α chain in the first subunit and the variable region and constant region of the T cell receptor β chain in the second subunit constitute the T cell receptor domain for recognizing the antigen peptide-HLA complex; The IgG antibody is a human IgG antibody; The anti-CD3 antibody is an anti-CD3ε antibody; The two CH3s in the second and third subunits were designed into knob and hole structures respectively using the knob-into-hole technique; LALA-PG mutations were introduced into the CH2 domains of the second and third subunits; The amino acid sequence of the first subunit is shown in SEQ ID No.20, the amino acid sequence of the second subunit is shown in SEQ ID No.21, the amino acid sequence of the third subunit is shown in SEQ ID No.22, and the amino acid sequence of the fourth subunit is shown in SEQ ID No.23. 2. A gene encoding the trispecific T cell engager according to claim 1, characterized in that the nucleotide sequence of the first subunit is shown in SEQ ID No.6, the nucleotide sequence of the second subunit is shown in SEQ ID No.7, the nucleotide sequence of the third subunit is shown in SEQ ID No.8, and the nucleotide sequence of the fourth subunit is shown in SEQ ID No.9. 3. Use of the trispecific T cell engager as described in claim 1 in the preparation of anti-tumor drugs, wherein the tumor cell type is NY-ESO-1SLL/HLA-A*02:01 and EGFR double-positive melanoma cells. 4. Use of the gene as claimed in claim 2 in the preparation of an anti-tumor drug, wherein the tumor cell type is a melanoma cell that is double positive for NY-ESO-1SLL/HLA-A*02:01 and EGFR. 5. An anti-tumor drug, characterized in that the active ingredient is the trispecific T cell engager according to claim 1 or the gene according to claim 2.