CN113912733A

CN113912733A - Immunotoxin targeting PD-L1 and preparation method and application thereof

Info

Publication number: CN113912733A
Application number: CN202010645801.9A
Authority: CN
Inventors: 谢捷明; 张彩云
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2020-07-07
Filing date: 2020-07-07
Publication date: 2022-01-11

Abstract

The invention relates to an immunotoxin targeting PD-L1, a preparation method and application thereof, wherein the immunotoxin comprises a carrier and a toxic molecule. The carrier is an antibody or other polypeptide capable of binding to the PD-L1 molecule; the toxic molecules are bacterial toxins, plant toxins and human-derived toxic molecules, and mutants of these toxins. The immunotoxin is the above toxic moleculeCoupling the carrier which can be combined with tumor cell PD-L1 into immunotoxin by a chemical cross-linking method, or expressing the immunotoxin by gene fusion of the two by a genetic engineering method; the immunotoxin can be used for treating tumor. The invention successfully prepares the CUS based on the pumpkin protein mutant_245CChemically conjugated immunotoxin D-CUS with PD-L1 monoclonal antibody Durvalumab_245C(ii) a The immunotoxin has obvious targeted killing effect on tumor cells over-expressed by PD-L1, and the maximum targeted therapeutic index can reach more than 110 ten thousand. Therefore, the immunotoxin targeting PD-L1 has wide anti-tumor application prospect.

Description

Immunotoxin targeting PD-L1 and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicines, and particularly relates to an immunotoxin targeting PD-L1, and a preparation method and application thereof.

Background

The immunotoxin is a hybrid molecule with specific cell killing capability, which is formed by coupling carrier molecules (such as antibodies, cytokines and the like) with guiding capability and toxic molecules (such as plant toxins, bacterial toxins, human protein toxins and the like) with cytotoxic effect.

Programmed cell death-ligand 1 (PD-L1) is a type I transmembrane protein of B7 family, and is expressed on the surfaces of various cancer cells and partial immune cells. After the PD-L1 on the cancer cell is combined with Programmed cell death receptor-1 (PD-1) on the T cell, T cell dysfunction, exhaustion and apoptosis can be caused, thereby inhibiting T cell mediated cell killing and leading the tumor cell to escape immune surveillance. The monoclonal antibody is used for blocking the interaction between PD-1 and PD-L1, and can destroy the PD-1 axis, thereby reversing the inhibiting effect of T cells, enhancing the endogenous anti-tumor immunity and showing good clinical effect. However, the patient's response rate to treatment with PD-1 or PD-L1 antibody was only 15% -30%. Even in melanoma, which is the most sensitive to immunotherapy, 60% of patients receiving PD-1/PD-L1 blocking treatment show primary resistance^[7]. Moreover, most patients will eventually develop acquired resistance over time after an initial response to the PD-1/PD-L1 blocker, leading to disease progression or relapse. Therefore, how to expand the clinical treatment scope and effect of PD-L1 immune checkpoint treatment becomes a new challenge for cancer treatment.

PD-L1 is expressed on the surface of melanoma, lung cancer, breast cancer, colon cancer and other cancer cells as a transmembrane glycoprotein. No expression of PD-L1 was detected in normal parenchymal tissues of the human body, including lung, liver, kidney, breast, colon, pancreas, uterus, and skeletal muscle. The immunotoxin targeting PD-L1 prepared by connecting a vector (such as an antibody including a complete antibody or a small molecule antibody) combined with PD-L1 and a toxic molecule is expected to kill tumor cells over-expressed by PD-L1 in a targeted manner, and overcomes the drug resistance problem of a PD-1/PD-L1 blocking agent.

Disclosure of Invention

The invention aims to provide a chemical crosslinking or genetic engineering method for preparing immunotoxin by connecting a carrier (an antibody or other polypeptide molecules combined with PD-L1) and toxic molecules (phytotoxin, bacterial toxin and human protein toxin); and tested for immunotoxin use.

The purpose of the invention is realized as follows:

the immunotoxin comprises toxic molecules and a carrier, wherein the toxic molecules are plant toxins, bacterial toxins, human protein toxins and mutants of the toxins; and immunotoxins were tested for anti-tumor activity.

Wherein, the immunotoxin is formed by coupling a toxic molecule or a mutant with an antibody of PD-L1 capable of binding tumor cells by a chemical crosslinking method to form the immunotoxin, or by gene fusion expression of the immunotoxin and the immunotoxin by a genetic engineering method;

specific pumpkin protein mutant CUS_245C(seq. ID no1) was conjugated with PD-L1 monoclonal antibody Durvalumab by chemical cross-linking method to prepare immunotoxin D-CUS_245CAnd used to determine its targeted killing effect on PD-L1 overexpressing tumors. The specificity of immunotoxin taking the pumpkin protein mutant as toxin molecule to tumor cells over-expressed by PD-L1 is enhanced, the activity of killing target cells is obviously improved, the use amount can be obviously reduced, and the toxicity is greatly reduced. In a word, the immunotoxin targeting PD-L1 taking the pumpkin protein mutant as the toxin molecule has the advantages of synergy and attenuation, and has wide application prospect.

Drawings

FIG. 1 immunofluorescence assay for Durvalumab and CUS_245CInternalization profiles in different tumor cells (a) levels of internalization by Durvalumab at different time points in response to PD-L1/SPC-A-1; (b) durvalumab does not act simultaneously with NC/SPC-A-1The level of internalization of the internodal points; (c) CUS_245CInternalization levels of 24h were effected with PD-L1/SPC-A-1 and NC/SPC-A-1 cells, respectively. Cells were incubated with FITC-labeled Durvalumab (green). Nuclei were counterstained with Hoechst (blue). Scale bar: 10 μm.

FIG. 2 SDS-PAGE analysis of the cross-linked and purified process product (a) and (b) are SDS-PAGE of 6% and 12% gels, respectively, under non-reducing conditions. M. protein Marker; 1. dimeric form of CUS_245C(ii) a 2. Monomeric CUS after DTT_245C(ii) a Durvalumab; crosslinking the resulting mixture; 5. ni warp²⁺A purified product; 6. final purified product (D-CUS) after dialysis_245C)

FIG. 3SRB method for detecting D-CUS_245CProliferation inhibition of different tumor cells (a) CUS_245C，D-CUS_245CD and D + CUS_245CDose-effect curve chart of action 72 h; (b) CUS_245C，D-CUS_245CD and D + CUS_245CDose-effect curve chart of 120h action.

FIG. 4 immunotoxin D-CUS_245CThe in vivo anti-tumor effect of the compound is to establish a nude mouse subcutaneous PD-L1/SPC-A-1 tumor-bearing model, and to inject 0.8mg/kg Durvalumab (D) and 0.4mg/kg CUS respectively through tail vein on the 4 th, 8 th, 12 th and 16 th days after grouping_245C，0.4mg/kg IT (D-CUS_245C-1) and 0.8mg/kg IT (D-CUS)_245C-2). (a) Tumor growth curves at different times for each group; (b) tumor weight; (c) graphs of the denuded transplants from each group; (d) body weight change before and after treatment, black bars (before treatment), gray bars (7 days after final treatment). Data are presented as mean ± SEM. P<0.01， ***P<0.001，****P<0.0001vs PBS。

Detailed Description

The invention is described in detail below with reference to the drawings and examples of the specification:

example 1: fluorescent labeling Durvalumab tracer combined PD-L1 and internalization process thereof

The method comprises the following steps: a human lung cancer cell line PD-L1/SPC-A-1 and a parental cell line SPC-A-1 (designated herein as NC/SPC-A-1 cell line) transfected with PD-L1 in logarithmic growth phase were taken. After digestion and counting, the cells were treated with 10% FBS-containing RPMI1640 complete medium diluted to 5X 10⁴Adding each cell per ml into a glass culture dish special for confocal focusing, adding 1ml of cells into each hole, placing at 37 ℃ and 5% CO₂Culturing in the incubator of (1) for 24 hours, sucking the cell culture medium in the culture dish, and treating the CUS with RPMI 1640_245CFITC and Durvalumab-FITC were diluted to 5ng/ml of treated cells, and after 1h, 2h, 6h, 12h and 24h of cell treatment, the well contents were discarded, and internalization was stopped with pre-cooled PBS. PBS in the wells is discarded, washed with precooled PBS for 3 times, Hoechst is diluted into 1 × working solution according to the instruction, incubated at room temperature of 1 ml/well for 15min and washed with PBS for 3 times, and finally 0.5ml of precooled PBS is added into the culture dish and protected from light. Cytofluorescence images were collected with FV500-IX81 confocal microscope (Olympus America inc., Melville, NY) and viewed with a 64-fold oil lens (NA ═ 1.40, Olympus, Melville, NY). Excitation and emission filters were used as alexa 488,488nm excitation, BP 520 ± 12nm emission filters; alexa 633,633nm excitation, LP650 color filter for emission; TAMRA,543nm excitation, emission with BP580 + -20 nm color filter. All experiments were repeated three times and analyzed with Leica software.

As a result: FIG. 1a shows that after 1 hour incubation of Durvalumab-FITC with PD-L1/SPC-A1 cells, green fluorescence stayed on the cell membrane surface, but not in the cytoplasm; at 2 hours, partial fluorescence was observed in the cytoplasm, but most of the fluorescence was still distributed on the membrane surface; at 6 hours the green fluorescence on the cell surface was reduced and a granular spot of concentrated and aggregated green fluorescence appeared in the cytoplasm. After 12 hours of treatment with Durvalumab-FITC, no green fluorescence was substantially visible on the cell surface, with a large number of green spots distributed in the cytoplasm; after 24 hours, the granular light spots were mainly concentrated in the near nucleus. When no PD-L1 was expressed, no fluorescence signal was detected both intracellularly and extracellularly using Durvallub-FITC for the same time as NC/SPC-A-1 cells (FIG. 1 b). The result shows that Durvalumab enters cells by virtue of receptor endocytosis and has high efficiency and specificity; CUS alone_245CRarely enter the cell.

The results show that the PD-L1 expressed on the surface of the tumor cells has obvious endocytosis.

Example 2: durvalumab and pumpkin protein mutant CUS_245CCoupling preparation of immunotoxin D-CUS_245C

The method comprises the following steps: dialyzing 2ml Durvalumab (5mg/ml) with dialysis tube with molecular weight cutoff of 8-10kDa, and changing PBS dialysate (1L for each PBS) every 3 hr for 5 times; after dialysis, 16.45. mu.l of a 20mmol/L solution of SPDP dissolved in DMSO was stirred at room temperature for 30min, and the reaction product (D-PDP) was dialyzed against the above buffer solution overnight to remove the excess SPDP. Another 3ml CUS_245C(total 7.45mg), adding appropriate amount of Dithiothreitol (DTT) dissolved with 0.01mol/L NaAc to make the final concentration of DTT 0.3mol/L, stirring at room temperature for reaction for 30min, dialyzing with 8-10kDa dialysis column, and removing excessive DTT by the same method as above; mixing Durvalumab after dialysis with CUS_245CMixing in a small beaker, stirring at 23 deg.C for 18h, adding appropriate amount of iodoacetamide solution to make final concentration 0.1mol/L, stirring at room temperature for 30min, centrifuging at 4 deg.C and 10000rpm for 10 min, collecting supernatant, dialyzing the supernatant with 100kDa dialysis tube, changing dialysate every 3h, 2000ml each time, and making total volume of dialysate be 10L, removing unconjugated CUS from the supernatant by dialysis method_245C(ii) a Diluting the dialyzed supernatant with 20mmol/L PB buffer solution 4-5 times, and loading on nickel ion affinity chromatography column (1ml nickel column medium) at a dropping speed of 0.3 ml/min; the equilibration column was eluted with about 30ml of 20mmol/L PB buffer (drop rate 2 ml/min); eluting 20ml (dropping speed is 0.3ml/min) of 15mmol/L imidazole eluent by tubes, collecting about 1.2ml of eluent in each EP tube, collecting 16 tubes, and removing unconjugated Durvalumab and a small amount of immunotoxin by the step; eluting with 150mmol/L imidazole, and receiving eluate at 8 EP tubes (dropping rate of 0.3ml/min), wherein each tube receives about 1.2ml imidazole, and high concentration imidazole can elute successfully coupled immunotoxin; sampling SDS-PAGE from each tube, collecting high-concentration sample tubes according to electrophoresis results, filtering, subpackaging, quantifying BCA, and measuring D-CUS_245CYield.

As a result: the electrophoresis of the purified protein is shown in FIG. 2, lane 4 of a and b, and the cross-linked products are Durvalumab and CUS_245CAccording to the following steps: 2,1: 3 to 1: 4 ratio of coupled mixture with multiple molecular weight, and free CUS_245CAnd do notThe reaction was complete in Durvalumab. Lane 5 of FIG. 2 shows that free CUS can be completely removed by dialysis_245C. The results show that the method is used for successfully preparing the coupled immunotoxin D-CUS. D-CUS_245CYield of (a): 10mg of Durvalumab and 7.45mg of CUS were used_245CReacting, purifying immunotoxin D-CUS_245CThe total yield (average molecular weight 230kDa) was 5.4mg, yielding (5.4/230)/(10/158) × 100% ═ 37.1%.

The above results indicate that the coupling immunotoxin D-CUS was successfully prepared by a chemical method_245C。

Example 3: immunotoxin D-CUS_245CIn vitro anti-tumor Activity assay

The method comprises the following steps: the PD-L1 over-expression human breast cancer cell line MDA-MB-231 cells and the PD-L1 transfected human lung cancer cell line PD-L1/SPC-A-1 and the PD-L1 low-expression lung cancer cell line NC/SPC-A-1 in logarithmic growth phase are respectively taken and inoculated on a 96-well culture plate, and each well is respectively inoculated with 4000/100 mu L. After 24h of culture, the experimental groups were supplemented with immunotoxins (D-CUS) at different concentrations_245C) Pumpkin protein mutant (CUS)_245C) Durvalumab (D) and mixture of pumpkin protein and Durvalumab with corresponding concentration (D + CUS)_245C)100 μ L, 3 duplicate wells per concentration; and adding 100 mu L of nutrient solution into the negative control group, and adding 200 mu L of nutrient solution into the blank control hole for zero setting of the instrument. After 72h or 120h, removing the supernatant, adding 100 mu L of precooled 10% trichloroacetic acid into each hole, and fixing for 1h at 4 ℃; pouring out the fixing liquid, washing the small holes with deionized water for 3 times, spin-drying, and air-drying; adding 50uL of SRB solution (Sulforhodamine B, 0.4% solution with 1% acetic acid) into each well, standing at room temperature for 30min, washing with 1% acetic acid solution for 4 times, and air drying; dissolving with 100uL 10mmol/L unbuffered Tris alkali solution (pH 10.5), and measuring absorbance (A) of each well at a wavelength of 570nm with a microplate reader (BIO-RAD product)₅₇₀) Taking the average value of each group, calculating the inhibition rate: inhibition rate ═ 1 (experimental group A)₅₇₀Control group A₅₇₀) X 100%. Statistical analysis with SPSS12.0 and calculation of IC₅₀. The experiment was repeated 2 times.

As a result: as can be seen from FIG. 3, the CUS alone_245CFor the above three cell lines at high concentration (>10 nmol/L) and is concentration-dependent. 1000nmol/L CUS_245CThe inhibition rates of the cells for 72 hours are respectively 68%, 52% and 68%, IC₅₀From 350nmol/L to 995 nmol/L; the inhibition rates at 120 hours were 78%, 74% and 93%, respectively, IC₅₀From 150nmol/L to 375 nmol/L. In contrast, D-CUS_245CCan inhibit cell proliferation at very low concentration. 0.1nmol/L of D-CUS_245CAfter 72 hours of action, the inhibition rates of PD-L1/SPC-A-1 cells and MDA-MB-231 cells are respectively 85 percent and 76 percent, and the IC is₅₀The drug effect ratio of the compound is (3.8 +/-1.27) pmol/L and (1.6 +/-0.11) pmol/L respectively_245C95526 to 314375 times stronger. When the time is extended to 120 hours, the drug effect is further improved. 0.1nmol/L of D-CUS_245CCan inhibit PD-L1/SPC-A-1 by 97% and MDA-MB-231, IC by 94%₅₀(2.8. + -. 0.081) pmol/L and (0.14. + -. 0.0075) pmol/L respectively, which indicates that D-CUS_245CNot only dose-but also time-dependent cytotoxic effects of (a). Durvalumab and D + CUS alone at the same concentration_245CHas no obvious effect. Whereas the effect on the PD-L1 low expression cell line NC/SPC-A-1 was on Durvalumab (D) and D + CUS at the same concentration gradient_245CIn contrast, D-CUS_245CDid not increase cytotoxicity (figure 3, table 1). After the maximum test concentration (1nmol/L) acts for 72h and 120h, the inhibition rate is 20 percent, and the IC is not reached₅₀The value is obtained.

The above results indicate that the immunotoxin D-CUS_245CHas obvious target killing effect on tumor cells over-expressed by PD-L1.

TABLE 1 immunotoxin D-CUS_245CIC of tumor cells acted for 72h and 120h₅₀(Mean±SD,n＝3)

Example 4: immunotoxin D-CUS_245CDetermination of anti-nude mouse transplantation tumor Activity

The method comprises the following steps: is taken at logarithmic growthRemoving old culture medium from long-term PD-L1/SPC-A-1 cells, washing with PBS, digesting with pancreatin, centrifuging, counting, and adjusting the concentration of cell suspension to 1 × 10 with serum-free RPMI 1640 culture medium⁸And/ml. The cell suspension was injected subcutaneously into the back of the right shoulder of nude mice with a 1ml sterile syringe, each 200. mu.l, 33 cells, and after injection, they were further kept in SPF-grade animal house, and the tumor growth was observed and the tumor volume was measured (V ═ 0.5 ab)²Wherein a is the major diameter and b is the minor diameter). Average volume of 50mm³On the left and right sides, they were divided into 5 groups (PBS, CUS) by the random number table method_245C、Durvalumab、 D-CUS_245CLow dose and D-CUS_245CHigh dose), 6-8 per group; PBS 200. mu.l, CUS was injected into each tail vein at 0, 4, 8 and 12 days after grouping, respectively_245C 0.4mg/kg、Durvalumab 0.8mg/kg、 D-CUS_245C0.4mg/kg and D-CUS_245C0.8mg/kg, and the body weight and the length and diameter of tumor mass of nude mice were measured at 0, 4, 6, 8, 12, 14, 16, 18, 20 and 22 days after administration, and the tumor volume was calculated. The tumor growth curve is drawn by taking the administration time as the abscissa and the tumor volume as the ordinate. Nude mice were sacrificed by cervical dislocation after 7 days after the last dose, subcutaneous tumors were completely detached, weighed and recorded. Calculating the tumor inhibition rate: tumor inhibition (IR%) (average tumor weight in negative control group-average tumor weight in experimental group)/average tumor weight in negative control group × 100%.

As a result: as shown in FIG. 4a, from day 4 to day 12, the injection was administered at 0.8mg/kg D-CUS_245CThe mice of the group showed a slow tumor growth with little increase in tumor size, while the other groups showed a gradual increase in tumor size, but the CUS_245CGroup sum D-CUS_245CThe low dose group (0.4mg/kg) showed slower tumor growth than Durvalumab and PBS groups. After day 14, tumor size began to increase in all treatment groups until day 22. But D-CUS_245CAnd CUS_245CTumors in the group grew much slower than those in the remaining two groups, and the difference was statistically significant (P)<0.05). On day 7 after the end of the administration, the mean tumor volume of the PBS group was greater than 1200mm³Nude mice were sacrificed by cervical dislocation, subcutaneous tumors were completely exfoliated, and the tumors of each group were weighed after removal of surface blood vessels. As shown in the figure4b and 4c, D-CUS_245CGroup mean tumor weight was significantly less than other groups (P)<0.01) and the higher the dosage, the more obvious the tumor inhibition effect. The tumor inhibition rate of the low-dose group was 55%, and that of the high-dose group was 67%. CUS_245CThe mean tumor weight of the group was also lower than that of the PBS group, but was not statistically significant, and the tumor inhibition rate was 32%. The tumor volumes and tumor weights of the Durvalumab group were not significantly different from those of the PBS control group (Table 2). Figure 4D shows that D-CUS is administered after 4 injections_245CAnd CUS_245CThe body weight of mice in all groups was reduced. However, all body mass index decreases were no more than 20% compared to pre-dose. During the experiment, no animal death occurred and all animals began to gain weight gradually after one week of withdrawal.

TABLE 2 immunotoxin D-CUS_245CInhibition effect on PD-L1/SPC-A-1 nude mouse transplantation tumor

**:VS.PBS,P<0.01；****:VS.PBS,P<0.0001

The above results indicate that the immunotoxin D-CUS_245CHas obvious inhibiting effect on nude mouse transplanted tumor of PD-L1 over-expressed tumor cell.

While the invention has been illustrated and described with respect to specific embodiments and alternatives thereof, it will be understood that various changes and modifications can be made without departing from the spirit and scope of the invention. It is understood, therefore, that the invention is not to be in any way limited except by the appended claims and their equivalents.

Sequence listing

<110> Fujian medical university

<120> immunotoxin targeting PD-L1 and preparation method and application thereof

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Asn Val Arg Phe Asp Leu Ser Ser Ala Thr Ser Ser Ser Tyr Lys Thr

1 5 10 15

Phe Ile Lys Asn Leu Arg Glu Ala Leu Pro Lys Asp Gly Lys Val Tyr

20 25 30

Asp Ile Pro Val Leu Leu Ser Thr Val Met Asp Ser Arg Arg Phe Ile

35 40 45

Leu Ile Asp Leu Val Asn Tyr Asp Gly Gln Ser Ile Thr Ala Ala Ile

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Asp Val Leu Asn Val Tyr Ile Val Ala Tyr Ser Thr Gly Thr Val Ser

65 70 75 80

Tyr Phe Phe Gln Gln Val Pro Ala Gln Ala Pro Lys Leu Leu Phe Lys

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Gly Thr Gln Gln Arg Thr Leu Pro Tyr Thr Gly Asn Tyr Glu Asn Leu

100 105 110

Gln Thr Ala Ala Lys Lys Leu Arg Glu Asn Ile Glu Leu Gly Leu Pro

115 120 125

Ala Leu Asp Ser Ala Ile Thr Thr Leu Phe His Tyr Asn Ala Glu Ala

130 135 140

Ala Ala Ser Ala Leu Leu Val Leu Ile Gln Thr Thr Ser Glu Ala Ala

145 150 155 160

Arg Phe Arg Tyr Ile Glu Leu Gln Ile Ala Asn Asn Val Gly Thr Lys

165 170 175

Phe Lys Pro Ser Gln Thr Ile Ile Ser Leu Glu Asn Asn Trp Ser Ala

180 185 190

Leu Ser Lys Gln Ile Gln Ile Ala Lys Asn Lys Asn Gly Gln Phe Glu

195 200 205

Thr Pro Val Ile Leu Ile Asp Pro Gln Gly Asn Arg Val Gln Ile Thr

210 215 220

Asn Val Thr Ser Asn Val Val Thr Gln Asn Ile Lys Leu Leu Leu Asn

225 230 235 240

Ile Gly Ala Thr Cys

245

Claims

1. An immunotoxin targeting PD-L1, characterized by: the immunotoxin comprises a carrier molecule capable of binding to PD-L1; and toxic molecules.

2. The immunotoxin targeting PD-L1 according to claim 1, characterized in that: the carrier molecule is an antibody molecule or polypeptide which can be combined with a PD-L1 molecule, wherein the antibody molecule comprises a complete antibody and a small molecule antibody, and specifically comprises Fab, ScFv, dsFv and a nano antibody.

3. The immunotoxin targeting PD-L1 according to claim 1, characterized in that: the toxic molecule is bacterial toxin, plant toxin or human-derived toxic molecule, and mutant of bacterial toxin, plant toxin or human-derived toxic molecule.

4. The immunotoxin targeting PD-L1 according to claim 3, characterized in that: the bacterial toxin is toxin derived from bacteria, including diphtheria toxin and pseudomonas aeruginosa exotoxin; the phytotoxin is plant ribosome-inactivating protein, including I type ribosome-inactivating protein including cucurbitacin, saporin, leukotoxin, trichosanthin, dianthus chinensis, luffa and balsam pear protein, and II type ribosome-inactivating protein including ricin and abrin and II type ribosome-inactivating protein A chain; the human protein toxin comprises RNA enzyme and apoptosis-promoting protein, wherein the apoptosis-promoting protein comprises Bcl-2 family protein, DNA fragmentation factor 40 and granzyme.

5. The method for producing an immunotoxin according to any one of claims 1 to 4, wherein: the toxin molecule or mutant thereof is chemically coupled to the carrier to form an immunotoxin.

6. The method for producing an immunotoxin according to any one of claims 1 to 4, wherein: through gene engineering means, the gene of the toxin molecule or the mutant thereof is fused with a carrier gene and the recombinant immunotoxin is expressed.

7. Use of an immunotoxin according to any one of claims 1 to 4: can be used for treating tumor.