CN116239700A - Tumor dual-targeting trispecific T cell adapter and application thereof - Google Patents

Abstract

The invention discloses preparation and application of a tumor double-targeting trispecific T cell adapter, and relates to the technical field of biological pharmacy. The tumor double-targeting trispecific T cell adapter has better specific T cell activation, proliferation activity and T cell anti-tumor activity on the cellular level, can be more effectively gathered in the inside of solid tumors, and is beneficial to the treatment of the solid tumors, so that the trispecific T cell adapter or the gene coded by the trispecific T cell adapter can be used for preparing anti-tumor drugs. The preparation method of the tumor double-targeting trispecific T cell adapter has the advantages of simple preparation and higher expression level.

Description

A tumor dual-targeting tri-specific T cell engager and its application

technical field

The invention relates to the technical field of biopharmaceuticals, in particular to the preparation and application of a tumor dual-targeting tri-specific T cell engager.

Background technique

Cancer has become an important disease that endangers human health. In recent years, T cell engagers have become one of the popular antibody anti-tumor drugs, showing good anti-tumor effects. Its mechanism of action is mainly to form a "tumor cell-T cell adapter-T cell" immune synapse by targeting tumor antigens and T cell CD3 domains, mediate T cells to participate in the induction of downstream signal transduction events, and activate T cells. Toxic mechanisms (including cytokine release, etc.), leading to tumor cell death.

Target selection is one of the key factors for the success of T cell engagers in treating tumors. At present, the targets of T cell engagers are mainly limited to the extracellular region of membrane proteins, not only the selection range is very limited (membrane proteins account for ~8% of total cell proteins). %), and the specificity is not strong. Therefore, the discovery 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 peptide-HLA complexes (pHLA), which are recognized by T cell receptors ( T-cell receptor, TCR) recognition.

Currently, TCR therapy can target intracellular and extracellular antigens, but this antigen peptide fragment (antigen peptide) must be presented on tumor cells by binding to a specific pHLA complex. Rare, high TCR-affinity pHLA complexes were the primary targets of study.

Because pHLA has a wide range of target selection and strong specificity, and TCR has a high degree of specificity in recognizing pHLA due to thymus selection, TCR-based T cell engagers are potential tumor immunotherapy drugs. However, the expression level of pHLA is extremely low, and the number of specific pHLA presented by a single cell ranges from several to several thousand, which is far lower than the high expression of antigens on the surface of traditional tumors (tens of thousands to hundreds of thousands), which greatly affects TCR-based T cells In vivo targeting and antitumor activity of the adapter. The trispecific T cell engager targeting pHLA and traditional tumor surface highly expressed antigens has both the characteristics of 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 many problems need to be solved urgently. Therefore, a TCR-based tumor dual-targeting T cell engager with good specificity needs to be developed.

Contents of the invention

The invention provides a preparation method and application of a TCR-based trispecific T cell engager with TCR and EGFR as target antigens.

Concrete 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, the framework comprising two heavy chains and two light chains, and the heavy chain comprising a heavy chain constant region CH1, CH2, CH3, the light chain includes CL, and the tumor dual-targeting trispecific T cell engager is a heterotetramer in any of the following forms:

(1) The first subunit includes the variable region, constant region and a light chain in the framework of T cell receptor α chain, and the second subunit includes the variable region, constant region and One heavy chain in the backbone, the third subunit includes the heavy chain variable region of the anti-EGFR antibody and another heavy chain in the backbone, and the fourth subunit includes the light chain variable region of the anti-EGFR antibody and the Another light chain in the backbone; 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; A heavy chain variable region and a light chain variable region of an anti-CD3 antibody are also connected to the first subunit or the fourth subunit;

(2) The first subunit includes the variable region, constant region and a light chain in the framework of T cell receptor α chain, and the second subunit includes the variable region, constant region and One heavy chain in the backbone, the third subunit includes the heavy chain variable region of the anti-CD3 antibody and another heavy chain in the backbone, and the fourth subunit includes the light chain variable region of the anti-CD3 antibody and the Another light chain in the backbone; 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 an anti-CD3 antibody domain; the backbone The heavy chain variable region and the light chain variable region of the anti-EGFR antibody are also connected to the two heavy chains in

In (1) and (2) above, 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 T cell receptor domain for recognition of antigenic peptide-HLA complexes. The IgG antibody is a human IgG antibody. The heavy chain includes constant regions CH1, CH2, CH3, and a hinge region in the constant regions.

The first subunit and the second subunit of the trispecific T cell adapter of the present invention contain a T cell receptor for recognizing the antigen peptide-HLA complex, and the antigen peptide is degraded by a tumor mutant protein in the proteasome, At the same time, it contains anti-EGFR antibody, a tri-specific T cell engager targeting pHLA and traditional tumor surface highly expressed antigens, and then uses anti-CD3 antibody to target the CD3 domain of T cells to tumor antigens, mediating T cells to participate in the induction of downstream signals Conducting events, activating the cytotoxic mechanism of T cells (including cytokine release, etc.), the results of the examples of the present invention show that the tumor dual-targeting tri-specific T cell engager has better specificity of T cells at the cellular level Activation, proliferation activity and anti-tumor activity of T cells can effectively lead to 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, and in application, all anti-EGFR antibodies are acceptable. The anti-CD3 antibody is an anti-CD3e antibody. But not limited to this.

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

Specifically, when the trispecific T cell adapter is (1) and the heavy chain variable region and the light chain variable region of the anti-CD3 antibody are connected to the first subunit, the amino acid sequence of the first subunit is as follows: 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 shown;

When the trispecific T cell adapter is (2) and a heavy chain in the backbone of the second subunit is connected with a heavy chain variable region and a light chain variable region of an anti-EGFR antibody, the first subunit The amino acid sequence of the second 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 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 heavy chain variable region of the anti-CD3 antibody is connected to the first subunit and For the light chain variable region, 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, and the nucleotide sequence of the third subunit The sequence is shown in SEQ ID No.8, and the nucleotide sequence of the fourth subunit is shown in SEQ ID No.9; when the three-specific T cell adapter is (2) and the backbone of the second subunit is When the heavy chain variable region and the light chain variable region of an anti-EGFR antibody are connected to a heavy chain of the first subunit, the nucleotide sequence of the first subunit is shown in SEQ ID No.1, and the nucleotide sequence of the second subunit The sequence is shown in SEQ ID No.2, the nucleotide sequence of the third subunit is shown in SEQ ID No.3, and the nucleotide sequence of the fourth subunit is shown in 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 encoding the first subunit, a gene encoding the second subunit, a gene encoding the third subunit, and The coding gene carrier of the fourth subunit; (2) the coding gene carrier of step (1) 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 carrier of the fourth subunit Transfect into mammalian cells, perform protein purification after culturing, and obtain the trispecific T cell engager. Preferably, the vector in step (1) is pLVX-Puro; the mammalian cell in step (2) is 293F cells. The present invention also provides the application of the trispecific T cell engager in the preparation of antitumor drugs. The invention also provides the application of the gene in the preparation of antitumor drugs. The present invention also provides an antitumor drug, the active ingredient of which is the trispecific T cell engager 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 gather in Inside solid tumors, it 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 make antitumor 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 of drawings

Figure 1 is a diagram of the molecular format of a tumor dual-targeting trispecific T cell engager and its control molecule.

Figure 2 is a graph showing the deglycosylation analysis of the tumor dual targeting trispecific T cell engager and its control molecule. Among them, (a) Deglycosylation analysis of tNY-aEGFR/aCD3 and tNY/aCD3, Lane 1: tNY/aCD3; Lane2: tNY/aCD3 treated with PNGase; Lane 3: tNY-aEGFR/aCD3; Lane 4: PNGase-treated tNY-aEGFR/aCD3; (b) Deglycosylation analysis of tNY-aCD3/aEGFR and control molecules; Lane 1: PNGase-treated tNY-aCD3/aEGFR; Lane2: tNY-aCD3/aEGFR; Lane 3 : PNGase-treated tCrtl-aCD3/aEGFR; Lane 4: tCrtl-aCD3/aEGFR; Lane 5: PNGase-treated tNY-aCD3/aCtrl; Lane 6: tNY-aCD3/aCtrl; Lane 7: PNGase; Lane 8: tNY/ aEGFR; Lane 9: PNGase-treated tNY/aEGFR.

Figure 3 is a graph showing 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) tNY-aEGFR/aCD3 and tNY/aCD3 to NY-ESO-1 SLL /HLA-A*02:01 specific affinity analysis; (c) affinity analysis of tNY-aCD3/aEGFR and its control molecule to NY-ESO-1 SLL/HLA-A*02:01; (d) tNY- Specific affinity analysis of aCD3/aEGFR and its control molecule to NY-ESO-1 SLL/HLA-A*02:01.

Fig. 4 is the affinity analysis of 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 molecule to PC-9 cells.

Fig. 5 is an analysis of the affinity 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-targeting trispecific T cell engager mediates PBMC killing of A375-NY cells. (a) tNY-aEGFR/aCD3 and tNY/aCD3 mediated PBMC killing of A375-NY cells; (b) tNY-aCD3/aEGFR and its control molecule mediated PBMC killing of A375-NY cells.

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

Figure 8 shows the expansion of T cells stimulated by tNY-aCD3/aEGFR.

Figure 9 shows that tNY-aCD3/aEGFR stimulates T cells to release gamma interferon.

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

Detailed ways

The animal experiments related to this application have been approved by the Experimental Animal Welfare Ethics Review Committee of Zhejiang University, and the application number is 12435. The relevant experiments involving humans in this application have been approved by the Medical Ethics Committee of the School of Pharmacy, Zhejiang University, and the approval document number is: (2018) Lunyan Pi No. 003.

Example 1

Preparation of a trispecific T cell engager for tumor dual targeting.

TCR selects NY-TCR (TCR targeting SLLMWITQC polypeptide and HLA-A*0201 complex, referred to as tNY), from patent: US20110014169A1; anti-EGFR antibody selects cetuximab (abbreviated as aEGFR, light chain variable region nucleosides) The acid sequence is from NCBI, sequence number: KX138561, the nucleotide sequence of the heavy chain variable region is from NCBI, sequence number: KX138562.1); the anti-CD3 antibody is HXR32, from the patent: WO2012162067A2. Co-designed two different molecular forms of tumor dual-targeting trispecific T cell engagers (Fig. ), constructing 14 expression plasmids for human kidney epithelial cells (293F).

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

tNY/aCD3: tNYECDα-GS-CL-His ₆ (gene sequence shown in SEQ ID No.29, SEQ ID No.1 and SEQID No.32), tNYECDβ-GS-CH1-CH2-CH3 (knob) (gene The sequence is shown in SEQ ID No.29 and SEQ ID No.5), aCD3VH-CL-CH2-CH3 (hole) (gene sequence is shown in SEQ ID No.29 and SEQ ID No.3), aCD3VL-CH1-Flag ( The gene sequence is 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 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 shown in SEQ ID No.29 and SEQ ID No.9).

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

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

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

Among them, tNYECDα and tNYECDβ are divided into 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 of three GGGGS lengths, aEGFRVH and aEGFRVL are the heavy chain variable region and light chain variable region of anti-EGFR, respectively, aCtrlVH and aCtrlVL are the heavy chain variable region and light chain variable region of the control antibody, respectively The variable region, aCD3VH and aCD3VL are the heavy chain variable region and light chain variable region of the anti-CD3ε antibody respectively, His ₆ is a tag composed of 6 histidines for protein purification, and the Flag tag is a tag of 8 for protein purification. A tag composed of two amino acids DYKDDDDK, CL, CH1, CH2, and CH3 are all IgG1 structures, and the knob-into-hole technology is used to design the knob and hole structures between the two CH3s, and the LALA-PG mutation is introduced into CH2 to make the Fc Silencing, CD3VH-CL-CH2-CH3 (hole) subunit and CD3VL-CH1-Flag subunit are crossmab structures.

M1亲和凝胶(购自Sigma公司，货号：A4596)进行柱层析纯化，获得相应蛋白。其中tNY-aCD3/aEGFR的表达量(约3mg/L)远高于tNY-aEGFR/aCD3(表达量约300μg/L)。用PNGase(购自Sigma公司，货号：PP0201)对纯化产物进行去糖基化处理，再用SDS-PAGE分析(以未经PNGase处理的纯化产物为对照)。The α-chain and β-chain extracellular regions of tNY and tCtrl, the heavy-chain variable region and light-chain variable region of aEGFR, aCD3 and aCtrl, the heavy-chain constant region and kappa light-chain constant region of IgG1 antibodies were provided by Shanghai Sangon Biotechnology Co., Ltd. Ltd Synthetics. Through PCR and overlapping PCR, each segment of the gene 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 were introduced downstream Restriction site (GAATTC), connected to the pLVX-Puro plasmid by T4 ligase (the plasmid is also double-digested by XhoI and EcoRI). Afterwards, the recombinant plasmid was transiently transferred to 293F cells, and the HisTrap HP affinity column (purchased from GE, catalog number: 17-5248-02) and anti-

M1 affinity gel (purchased from Sigma, product number: A4596) was purified by column chromatography to obtain the corresponding protein. The expression level of tNY-aCD3/aEGFR (about 3mg/L) was much higher than that of tNY-aEGFR/aCD3 (about 300μg/L). The purified product was deglycosylated with PNGase (purchased from Sigma, product number: PP0201), and then analyzed by SDS-PAGE (the purified product without PNGase treatment was used as the control).

As shown in Figure 2, the subunits of the T cell adapter treated with PNGase all appeared at the positions of theoretical sizes (see Table 1 for the theoretical molecular weight of each subunit of the T cell adapter), in which the β chain and α chain of TCR The position changes before and after PNGase treatment, indicating that there is obvious glycosylation modification, the α chain changes more, and the glycosylation modification is more complex.

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

Example 2

(1) Affinity verification

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

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

100 μL of 1 μg/mL streptavidin (purchased from Shanghai Sangon Biotechnology Co., Ltd., catalog number: A100497) was coated in a 96-well EIA/RIA plate (purchased from Coming Company, catalog number: 03619013) overnight at 4°C. After washing 4 times with PBST (PBS solution containing 0.05% Tween 20), block at 37° C. for 1 hour (blocking solution: 5% skimmed milk powder in PBST solution). After washing with PBST for 4 times, incubate 100 μL of 1 μg/mL NY-ESO-1/A02-Biotin, WT1/A02-Biotin or Tax/A02-Biotin at 37°C for 1 hour. After washing with PBST for 4 times, 100 μL of tumor dual-targeting tri-specific T cell engager and control molecules with gradient concentration were incubated at 37°C for 1 h. After washing with PBST for 4 times, horseradish peroxidase-labeled goat anti-human IgG (H+L) (purchased from Beyond Biotechnology Co., Ltd., catalog number: A0201) was incubated at 37°C for 1 h. After washing with PBST for 5 times, 100 μL of LTMB liquid substrate (purchased from Sigma, product number: T4444) was added to develop color for about 10 min, and 100 μL of 2M sulfuric acid was added to stop 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 contained tNYECD subunits and thus had the effect on NY-ESO-1 SLL/HLA-A*02:01 High affinity, and it is concentration-dependent; while tCrtl-aCD3/aEGFR has no affinity for NY-ESO-1 SLL/HLA-A*02:01, which can be used as a control molecule in subsequent drug evaluation (Figure 3a, 3c ). tNY-aEGFR/aCD3, tNY/aCD3, tNY-aCD3/aEGFR, tNY-aCD3/aCtrl and tNY/aEGFR have no affinity for WT 1RMF/HLA-A*02:01 and TaxLLF/HLA-A*02:01, It shows that it has good affinity and specificity to NY-ESO-1SLL/HLA-A*02:01 (Fig. 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 incubated at 4°C in 200 μL respectively, with different concentrations of tumor dual-targeting trispecific T cell engagers and their control molecules for 30 minutes (with Incubate in PBS as a control). After washing with PBS twice, incubate at 4°C with 200 μL FITC-labeled goat anti-human IgG (H+L) (purchased from Beyond Biotechnology Co., Ltd., catalog number: A0556). After washing with PBS twice, the cells were resuspended with 400 μL PBS, and then detected by ACEA NovoCyteTM flow cytometer.

The results are shown in Figure 4a, the average fluorescence intensity of PC-9 cells incubated with tNY-aEGFR/aCD3 increased with the increase of concentration, indicating that tNY-aEGFR/aCD3 can recognize EGFR antigen in a concentration-dependent manner; tNY/aCD3 does not contain The aEGFR subunit cannot recognize EGFR antigen, so it has no affinity for PC-9 cells. The average fluorescence intensity of PC-9 cells incubated with tNY-aCD3/aEGFR, tCrtl-aCD3/aEGFR and tNY/aEGFR was significantly higher than that of tNY-aCD3/aCtrl in a concentration-dependent manner, indicating that tNY-aCD3/aEGFR, tCrtl- aCD3/aEGFR and tNY/aEGFR can recognize the EGFR antigen on the surface of PC-9 cells; tNY-aCD3/aCtrl does not contain aEGFR subunit, 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 incubated at 4°C in 200 μL respectively, with different concentrations of tumor dual-targeting tri-specific T cell engagers and their control molecules for 30 minutes (incubating PBS is the control). After washing with PBS twice, incubate at 4°C with 200 μL FITC-labeled goat anti-human IgG (H+L) (purchased from Beyond Biotechnology Co., Ltd., catalog number: A0556). After washing with PBS twice, the cells were resuspended with 400 μL PBS, and then detected by ACEA NovoCyteTM flow cytometer.

The results showed that the affinity of tNY-aEGFR/aCD3 and tNY/aCD3 to Jurkat cells was consistent and concentration-dependent (Fig. 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 in a concentration-dependent manner, indicating that tNY-aCD3/aEGFR 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: Specific antitumor activity in vitro

A375-NY cells (NY-ESO-1SLL/HLA-A*02:01 ⁺ and EGFR ⁺ ) stably transfected with EGFP and presenting polypeptide SLLMWITQC and PBMC (purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., Cat. No.: A10S045018) were spread on 24-well cell culture plates at a ratio of 1:4, and a series of gradient concentrations of tNY/aCD3/aEGFR or tNY/aCD3 (n=3) were added, mixed well and placed in a 37°C, 5% CO ₂ incubator for incubation . After 48 hours, the cells were collected, stained with Annexin V, 633 Apoptosis Detection Kit (purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., Cat. No.: AD11), and the apoptosis of tumor cells was detected by ACEA NovoCyteTM flow cytometer. The results are shown in Figure 6a. Although tNY-aEGFR/aCD3 can mediate PBMCs to kill A375-NY cells, the killing activity is not much different from that of tNY/aCD3, indicating that the molecular form of tNY-aEGFR/aCD3 does not mediate the generation of T cells. The form with the best antitumor activity.

A375-NY cells and PBMCs were spread in 96-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 placed at 37°C, 5% _CO2 incubator incubation. After 48 hours, the culture supernatant was collected, detected with a lactate dehydrogenase detection kit (purchased from Beyontian Biotechnology Co., Ltd., Cat. No.: C0O17), and the EC50 was calculated using GraphPad software. The results are shown in Figure 6b. tNY-aCD3/aEGFR showed a good killing activity in mediating PBMC on NY-ESO-1 SLL/HLA-A*02:01 and EGFR double-positive A375-NY cells, and the concentration was Dependence, the killing activity is much higher than tNY-aCD3/aCtrl. From the fact that tCrtl-aCD3/aEGFR and tNY/aEGFR only mediated the weak killing activity of PBMC under high concentration conditions, the specificity of tNY-aCD3/aEGFR was good.

Example 4: Detection of T cell specific activation

A375-NY cells and PBMCs were spread 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 placed at 37°C, 5% _CO2 incubator incubation.

After culturing for 72 h, cells were collected by centrifugation at 600×g for 5 min, washed once with 1×PBS, resuspended in 1×PBS, and 5 μLAPC-labeled mouse anti-human CD3 antibody (purchased from ThermoFisher Scientific, Cat. No. 17-0037- 42), 5 μL Super Bright 436-labeled mouse anti-human CD69 antibody (purchased from ThermoFisher Scientific, product number 62-0699-42) and 1 μL PE-labeled mouse anti-human CD25 antibody (purchased from ThermoFisher Scientific company, product number 12-0259- 80), incubate at 4°C for 30 minutes in the dark. After washing twice with 1×PBS, resuspend in 200 μL of 1×PBS, and use CytomicFC 500MCL flow cytometer to detect the ratio of CD69 and CD25 positive T cells (CD3 positive).

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 with tNY-aCD3/aEGFR and A375-NY can specifically and significantly activate T cells, and the activation ratio is much higher than that of various controls molecules, co-incubation of tNY-aCD3/aEGFR with A375-NY can significantly activate T cells even at a very low concentration of 20pM.

Example 5: Detection of T cell proliferation

Collect PBMCs by centrifugation at 600×g for 10 min, wash twice with 1×PBS preheated at room temperature, resuspend in 1×PBS, and add CFSE (purchased from ThermoFisher Scientific, catalog number: 65-0850-84) to the final concentration to 2.5μM, mix well, and incubate at 37°C for 15min in the dark. Collect A375-NY and CFSE-treated PBMCs by centrifugation at 600×g for 10 minutes. After washing once with PBS, spread A375-NY and CFSE-treated PBMCs in 48-well cell culture plates at a ratio of 1:4, and add a series of gradient concentrations of tNY - aCD3/aEGFR and its control molecules (n=3), mixed well and placed in a 5% CO ₂ incubator at 37° C. for incubation.

After culturing for 96 hours, collect the cells by centrifugation at 600×g for 5 minutes, wash twice with 1×PBS, resuspend in 100 μL of 1×PBS, add 5 μL of PI stock solution (1 mg/mL), incubate at room temperature for 15 minutes in the dark, then incubate at 600×g for 5 minutes Centrifuge, add 400 μL 1×PBS to resuspend, and use ACEA NovoCyteTM flow cytometer to select PBMC according to cell morphology to detect its proliferation.

After activation, T cells can divide and proliferate. CFSE can non-specifically bind to proteins in cells to produce green fluorescence, which is evenly distributed to the two progeny cells as the cell divides, and the green fluorescence gradually decreases with the increase in the number of divisions. The results are shown in Figure 8. Compared with the blank control group, co-incubation of tNY-aCD3/aEGFR and A375-NY can stimulate the proliferation of T cells, and the proliferation ratio is proportional to the concentration; compared with tNY-aCD3/aEGFR , tNY-aCD3/aCtrl, tCrtl-aCD3/aEGFR and tNY/aEGFR can stimulate T cell proliferation only when co-incubated with A375-NY at a high concentration of 2000pM, and the proliferation factor is much lower than tNY-aCD3/aEGFR, indicating that tNY- The co-incubation of aCD3/aEGFR and A375-NY stimulated the proliferation of T cells with strong and specificity.

Embodiment 6: Detection of gamma interferon

A375-NY cells and PBMCs were spread 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 placed at 37°C, 5% _CO2 incubator incubation.

After culturing for 72 hours, centrifuge at 600×g for 5 minutes to collect the supernatant, dilute to a certain concentration, and detect the release of interferon-γ by T cells with an interferon-γ detection kit (purchased from ThermoFisher Scientific, catalog number: 88-7316-88) Condition.

The results are shown in Figure 9. Interferon-γ is a cytokine released by T cells after activation, which has a strong tumor-inhibiting effect. Co-incubation of tNY-aCD3/aEGFR with A375-NY cells can significantly stimulate a large number of T cells. Release γ-interferon in a concentration-dependent manner; co-incubation of tNY-aCD3/aCtrl and A375-NY cells can also stimulate T cells to release γ-interferon, but the release amount is significantly lower than tNY-aCD3/aEGFR; tCrtl-aCD3/aEGFR Co-incubation with A375-NY cells can hardly stimulate T cells to release interferon-γ, 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 underarm of 8-week-old female SCID-Beige mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., SPF grade) according to the 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), 4 mice in each group, and administered via tail vein (100 μg/aEGFR group). kg, and the normal saline group was given an equal volume of PBS). Thereafter, the drug was administered every 3 days, for a total of 3 times. The tumor size of the mice 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 be effective at lower doses Inhibit the growth of xenografted tumors in mice with good specificity; compared with the normal saline group and the tCrtl-aCD3/aEGFR group, the tNY-aCD3/aCtrl group has a certain effect on inhibiting tumor growth, but the anti-tumor effect in vivo is significantly inferior to that of tNY - aCD3/aEGFR group.

Claims (10)

1. A tumor dual-targeting trispecific T cell engager, comprising a framework comprising the constant region of an IgG antibody, the framework comprising two heavy chains and two light chains, the heavy chain comprising the heavy chain constant region CH1, CH2, CH3, light chain including CL, characterized in that the tumor dual-targeting trispecific T cell engager is a heterotetramer, in any of the following forms: (1) The first subunit includes the variable region, constant region and a light chain in the framework of T cell receptor α chain, and the second subunit includes the variable region, constant region and One heavy chain in the backbone, the third subunit includes the heavy chain variable region of the anti-EGFR antibody and another heavy chain in the backbone, and the fourth subunit includes the light chain variable region of the anti-EGFR antibody and the Another light chain in the backbone; 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; A heavy chain variable region and a light chain variable region of an anti-CD3 antibody are also connected to the first subunit or the fourth subunit; (2) The first subunit includes the variable region, constant region and a light chain in the framework of T cell receptor α chain, and the second subunit includes the variable region, constant region and One heavy chain in the backbone, the third subunit includes the heavy chain variable region of the anti-CD3 antibody and another heavy chain in the backbone, and the fourth subunit includes the light chain variable region of the anti-CD3 antibody and the Another light chain in the backbone; 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 an anti-CD3 antibody domain; the backbone The heavy chain variable region and the light chain variable region of the anti-EGFR antibody are also connected to the two heavy chains in In (1) and (2) above, 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 T cell receptor domain for recognition of antigenic peptide-HLA complexes. 2. The trispecific T cell engager according to claim 1, wherein the IgG antibody is a human IgG antibody. 3. The trispecific T cell engager according to claim 1, wherein the anti-CD3 antibody is an anti-CD3ε antibody. 4. The trispecific T cell engager according to claim 1, wherein the two CH3s in the second subunit and the third subunit are designed into a knob structure and a hole using the knob-into-hole technology respectively. structure. 5. The trispecific T cell engager of claim 4, wherein the CH2 domains of the second subunit and the third subunit introduce LALA-PG mutations. 6. The trispecific T cell engager of claim 5, wherein When the trispecific T cell adapter is (1) and the heavy chain variable region and the light chain variable region of the anti-CD3 antibody are connected to the first subunit, the amino acid sequence of the first subunit is as 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 adapter is (2) and a heavy chain in the backbone of the second subunit is connected with a heavy chain variable region and a light chain variable region of an anti-EGFR antibody, the first subunit The amino acid sequence of the amino acid sequence 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 As shown in SEQ ID No.18. 7. The gene encoding the trispecific T cell engager according to any one of claims 1 to 6, when the trispecific T cell engager is (1) and the heavy chain of the anti-CD3 antibody is connected to the first subunit For the variable region and the light chain variable region, the nucleotide sequence of the first subunit is as shown in SEQ ID No.6, the nucleotide sequence of the second subunit is as shown in SEQ ID No.7, and the nucleotide sequence of the third subunit is as shown in SEQ ID No.7. The nucleotide sequence of the fourth subunit is shown in SEQ ID No.8, and the nucleotide sequence of the fourth subunit is shown in SEQ ID No.9; When the trispecific T cell adapter is (2) and a heavy chain in the backbone of the second subunit is connected with a heavy chain variable region and a light chain variable region of an anti-EGFR antibody, the first subunit The nucleotide sequence of the second subunit is shown in SEQ ID No.1, the nucleotide sequence of the second subunit is shown in SEQ ID No.2, the nucleotide sequence of the third subunit is shown in SEQ ID No.3, and the nucleotide sequence of the second subunit is shown in SEQ ID No.3. The nucleotide sequence of the tetrasubunit is shown in SEQ ID No.4. 8. The use of the trispecific T cell engager according to any one of claims 1 to 6 in the preparation of antitumor drugs. 9. The application of the gene as claimed in claim 7 in the preparation of antitumor drugs. 10 . An antitumor drug, characterized in that the active ingredient is the trispecific T cell engager according to any one of claims 1 to 6 or the gene according to claim 7 .

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