WO2017129065A1

WO2017129065A1 - Plasmid comprising targeted anti-cancer gene, and construction method and application thereof

Info

Publication number: WO2017129065A1
Application number: PCT/CN2017/071921
Authority: WO
Inventors: 钮利喜; 李卓玉; 石亚伟; 李佳悦; 肖虹; 石瑛
Original assignee: 山西大学
Priority date: 2016-01-30
Filing date: 2017-01-20
Publication date: 2017-08-03
Also published as: CN105734080A

Abstract

Disclosed are a plasmid comprising a targeted anti-cancer gene and a construction method and application thereof. The method comprises the following main steps: connecting a transcription promoter with a tumor-targeting property to a therapeutic gene, inserting the connected sequence into a eukaryotic-expression plasmid, and obtaining a recombinant expression plasmid carrying the therapeutic gene. The plasmid can be used in a PEI-mediated nebulization method, and is able to treat a lung tumor.

Description

[Name of invention established by ISA according to Rule 37.2] A plasmid comprising a targeted anti-cancer gene and a construction method and application thereof

The present application claims priority from Chinese Patent Application No. CN2016100670036, filed on Jan. 30, 2016. This application cites the entire text of the above-mentioned Chinese patent application.

Technical field

The present invention relates to gene therapy drugs, and in particular to a targeted anti-cancer gene-plasmid and its construction method and application.

Background technique

Gene therapy is a biotechnology program that has emerged in the past ten years. In the whole gene therapy, tumor gene therapy programs account for more than 60%. Gene therapy is considered to be the hope of humans to finally conquer tumors. Currently, it is divided into carriers for gene therapy. Both viral and non-viral types. Viral vectors include: adenovirus, adeno-associated virus, retrovirus, lentivirus, and herpes virus. The viral vector has a high transfection rate and a long expression time, but the immunogenicity is strong and there is a certain risk. Non-viral vectors include: naked DNA, DNA coated with transfection reagents. With the development of in vivo transfection methods and transfection reagents, non-viral vectors have attracted much attention because of their advantages of small immunogen, safety, ease of use, and large-scale production. In recent years, a new polycationic compound, polyethylenimine (PEI), provides an effective new technical approach for gene transfer. PEI molecules have polymers with different molecular weights and are transfected in vitro. Among them, it is agreed that the 25KD PEI transfection efficiency is higher and the toxicity is lower. At the working concentration (1.29～6.45μg/mL), the toxicity rating is 0～1. Since the PEI/DNA complex is structurally stable and easy to pass through the respiratory barrier, in recent years, gene transfer by atomization has become an effective way for respiratory gene therapy and immunity. For example, Nyce et al. treats asthma in mice by aerosol inhalation of antisense oligonucleotides (Nature, 385: 721-725, 1997, the entire disclosure of which is hereby incorporated by reference in its entirety) in the entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire all all all all all all all in all

Purpose of the invention

It is an object of the present invention to provide a recombinant gene-plasmid which is simple in operation, low in cost, and which has high anticancer activity and can be used for gene therapy operations.

Another object of the present invention is to provide a method of constructing a targeted anti-cancer gene-plasmid.

It is yet another object of the present invention to provide a use of a targeted anti-oncogene-plasmid for treating a tumor.

In order to achieve the above object, the technical solution of the present invention is:

A targeted anti-cancer gene-plasmid, which is a plasmid containing a tumor-specific promoter and carries a tumor therapeutic gene, which carries a tumor therapeutic gene and carries two different tumor therapeutic genes simultaneously Or carry a tumor treatment gene and a protease gene that can modify the processing of tumor expression gene expression products.

A method for constructing an anti-cancer gene-plasmid, comprising the steps of: a tumor-specific promoter, two tumor therapeutic genes or a tumor therapeutic gene, and a protease gene responsible for modifying a tumor therapeutic gene expression product in turn The cloned into the eukaryotic expression plasmid to obtain a targeted anti-oncogene-plasmid; the two genes are linked by a ribosome entry site.

Among them, tumor tissue-specific promoters include, but are not limited to, telomerase reverse transcriptase (TERT) promoter, alpha-fetoprotein (ATP) promoter, carcinoembryonic antigen (CEA) promoter, prostate-specific antigen promoter and Breast cancer tissue-specific promoter. The use of a tumor tissue-specific promoter allows the expression of an anti-oncogene to be specifically performed in tumor cells without performing in normal cells.

The tumor therapeutic genes include, but are not limited to, a TRAIL gene, a tumor suppressor gene, a cytokine gene, a pro-apoptotic gene, a toxin protein gene, an angiogenesis gene, a suicide gene, and other genes in the tumor necrosis factor superfamily.

The tumor suppressor gene is p53, PTEN, Rb, NF1, VHL or APC; the cytokine genes are IL-2, IL-12, IL-24, GM-CSF, INF-α, INF-β, INF- γ; the pro-apoptotic gene is TRAIL, Smac, Omi, Bax, Caspase-3, Caspase-7 or Eorf4; the toxin protein gene is melittin melittin, scorpion BMK, ribosome inactivating protein gelonin, Snail Toxin CTX; the suicide gene is cd or tk; the angiogenesis inhibitor gene is endostatin, angiostatin k1-4, angiostatin k1-3, plasminogen k5 or sflt- 1.

The protease genes include, but are not limited to, an enterokinase light chain gene, a thrombin gene, a factor Xa gene, a tobacco etch virus protease (TEV protease) gene, and a human rhinovirus. 3C protease (HRV 3C Protease) gene.

The targeted anti-cancer gene-plasmid constructed by the invention can be used for developing a new anticancer drug for treating tumors effectively, and can also be combined with other compounds to form a pharmaceutical composition, which can be: chemotherapeutic drugs, biotoxins, immunosuppression Compound, monoclonal antibody.

The beneficial effects of the invention:

1. The targeted anti-cancer gene-plasmid provided by the invention has been proved by cell experiments that the anti-oncogene can specifically express in tumor cells and induce tumor cell apoptosis. It has been proved by animal experiments that it can be used for the treatment of lung tumors by atomized gene therapy.

2. The present invention provides a method for constructing a recombinant eukaryotic expression plasmid carrying two anti-oncogenes or an anti-oncogene and a protease gene, which is simple and easy to grasp.

3. The gene-plasmid constructed by the present invention can specifically express the foreign gene carried in the tumor cell, and thus the gene-plasmid has a high targeted anticancer effect.

4. The targeted anti-cancer gene-plasmid constructed by the invention can be selectively killed in animal experiments without affecting normal cells, and the novel gene-plasmid targeted therapy can substantially eliminate the tumor. It has laid a good foundation for the future treatment of human cancer.

DRAWINGS

Figure 1 is a schematic representation of the gene-plasmid phTERTcp-hmel of the present invention

Figure 2 is a schematic representation of the gene-plasmid pmTERTp-hmel of the present invention

Figure 3 Results of induction of apoptosis after transfection of Hela cells with gene-plasmid phTERTcp-hmel

Figure 4 atomized gene therapy device

Figure 5 shows the mouse model of melanoma cell lung metastasis using gene-plasmid pmTERTp-hmel The results of the type of aerosol gene therapy were: control group (A), treatment group (B).

Figure 6 Comparison of pathological sections between treatment group (B) and control group (A)

detailed description

The invention is further illustrated by the following examples, which are intended to illustrate the invention and not to limit the scope of the invention.

Example 1 Construction of Targeting Anticancer Gene- Plasmid phTERTcp-hmel

Melittin, also known as bee hemolytic peptide, is one of the main components of Italian bee venom and is one of the most active active substances in bee venom. It plays a major pharmacological role in bee venom and has a high degree of Biological activity. Melittin accounts for 50% of the dry weight of bee venom and is easily soluble in water and is strongly alkaline (pH=10). Melittin can affect cell signaling pathway and induce apoptosis. It has antibacterial, anti-inflammatory and anti-radiation effects. In recent years, it has also been found to have special effects in anti-tumor and anti-HIV. However, because melittin has the same killing effect on mammalian normal cells, its clinical application is limited. The melittin is processed in the body three times (two cuts at the N-terminus and one cut at the C-terminus) to finally become a biologically active mature polypeptide, so the first amino acid residue at the N-terminus of the mature melittin is not Met. This entails considering in genetic engineering operations how to process a recombinant gene expression product into a polypeptide having exactly the same sequence as a natural melittin and to limit its expression to tumor cells. Therefore, we used the human telomerase reverse transcription core promoter (hTERTcp) to drive it, and added a start codon and a enterokinase recognition sequence (Met-Asp-Asp-Asp-Asp-Lys) at its N-terminus. The human enterokinase light chain protein (hEKL) was co-expressed by the ribosome entry site (IRES) and ligated by the enterokinase recognition sequence at its C-terminus, and a melittin gene was expressed and expressed to increase the target gene. The amount of expression. Through targeted transcription and simulation of natural processing, the recombinant melittin gene can express the polypeptide sequence completely consistent with the natural melittin sequence in the cell, greatly improving the cure rate of the tumor and eliminating the normal cells in the body. hurt.

The sequence of the melittin gene was synthesized by Nanjing Jinseli Company, and the start codon was added at the N-terminus. ATG and the enterokinase recognition sequence correspond to the coding sequence, and the codons of the gene are optimized according to human codon bias. The specific sequence (SEQ ID NO: 1) is as follows:

Design the following primers:

pUCUV5-hmel was purchased from Nanjing Kingsray Company and contains a synthetic melittin gene.

pUCUV5-hEKL was purchased from Nanjing Kingsray Company and contains human enterokinase light chain gene.

The pGL3-Enhancer plasmid was purchased from Promaga.

The pIRES plasmid was purchased from Clontech.

The whole genome DNA of HepG2 cells was extracted with genomic DNA kit, and the primers FW (hTERTcp) and RV (hTERTcp) were used for PCR amplification, and the 262 bp fragment was recovered by electrophoresis. The PCR product was digested with Sac I/Xba I and cloned into the same pGL3-Enhancer plasmid, and the new plasmid was named phTERTcp.

The plasmid pUCUV5-hmel was used as a template, and FW (hmel) and RV (hmel) 01 were used as primers for PCR amplification. The 96 bp fragment was recovered by electrophoresis, and the PCR product was digested with Nde I/Xhol I and cloned into the same enzyme. The phTERTcp plasmid was obtained, and the recombinant plasmid pGL3-hTERTcp-hmel was obtained.

Using plasmid pUCUV5-hEKL as template, PCR amplification with primer FW (hEKL) and primer RV (hEKL-hmel), PCR with plasmid pUCUV5-hmel as template and FW (hmel) and RV(hmel)02 as primers Amplification, using the above two PCR products as a template, primer-FW (hEKL) and primer RV (hmel) 02 for PCR amplification of the hEKL-hmel gene fragment; pIRES plasmid as a template, primer FW (IRES) and Primer RV (IRES) was amplified by PCR to obtain the IRES gene fragment, using IRES and hEKL-hmel as templates, with primer FW (IRES) and primer RV (hmel) 02 The gene fragment IRES-hEKL-hmel was amplified by bridge PCR, and the vector pGL3-hTERTp-hmel and the gene fragment IRES-hEKL-hmel were separately subjected to Xho I/Xba I double digestion and gel recovery, and T4 was used according to a certain ratio. The enzyme was ligated overnight at 16 °C to transform competent cells E.coli DH5α. The positive clones were screened by PCR first. Finally, the positive recombinants were further digested and sequenced to verify the results. The recombinant plasmid was named phTERTcp-hmel (Fig. 1).

Example 2 Construction of Targeting Anticancer Gene-plasmid pmTERTp-hmel

Since the mouse telomerase reverse transcriptase promoter sequence and the human telomerase reverse transcriptase promoter have low sequence homology, we replaced hTERTcp in the recombinant plasmid with mouse telomere in animal experiments. The enzyme reverse transcriptase promoter (mTERTp) was specifically operated as follows: 30 mg of the leg muscle of a normal mouse was taken, and the whole genome DNA of the mouse was extracted according to the instructions of the Biomiga genomic extraction kit. Then, the mouse genome was used as a template, and FW (mTERTp) 01 and RV (mTERTp) 02 were used for PCR amplification of the upstream and downstream primers to obtain a mTERTp gene fragment. The purified PCR product was recovered according to the DNA recovery kit instructions, and the resulting fragment of interest was ligated into the pEASY-T1 vector, designated pEASY-T1-mTERTp, and transformed into E. coli DH5α. After screening with blue and white spots, white colony extraction plasmids were picked and identified by double enzyme digestion. Positive transformants were verified by sequencing by Huada Gene Biotechnology Co., Ltd. Using pEASY-T1-mTERTp as a template and using FW(mNde I)01 and RV(mNde I)02 as primers, the Nde I restriction site in the mTERTp sequence was removed using a point mutation kit, and the mutant sequence was confirmed by sequencing. The mutated mTERTp fragment was excised from the pEASY-T1-mTERTp plasmid using Sac I and Nde I, phTERTcp-hmel Sac I and Nde I were double-digested and electrophoresed, and a large fragment was recovered by gel, and then ligated with the obtained mTERTp to obtain a recombinant plasmid pmTERTp-hmel (Fig. 2), which was confirmed by sequence measurement.

Example 3 Targeting Transcription Melittin Induces Hela Cell Apoptosis in Vitro

HeLa cells were routinely cultured in high glucose DMEM medium (containing 10% fetal bovine serum, 0.1 mg/mL streptomycin and 100 U/mL penicillin) under the culture conditions: humidified in a 37 ° C, 5% CO ₂ incubator. The cultured cells (2×10 ⁵ /well) were inoculated into a 6-well plate. When the cells were grown to a cell density of 60-70%, the recombinant plasmid phTERTcp-hmel was transiently transfected into HeLa cells using Lipofectamine 2000. The group was treated with only equal amounts of liposomes. After 48 hours of cell transfection, laser confocal scanning and photographing were performed. The results are shown in Figure 3. The typical characteristics of apoptosis in the experimental group were: adherent cells shrink, round, and fall off, and the rate of division is also slowed down.

Example 4 Gene therapy of mouse lung tumor model using targeted expression of melittin

A. Large-scale extraction of plasmid pmTERTp-hmel by alkaline lysis

1) The pmTERTp-hmel plasmid was transformed into E. coli DH5α, plated on LB solid medium with ampicillin resistance, and cultured overnight.

2) Pick a single colony in a 5 mL LB tube, shake culture at 37 ° C, then transfer to a 50 mL LB small flask and continue to shake culture at 37 ° C.

3) According to the 2% inoculation amount, the bacterial liquid was transferred to a 1000 mL LB flask and shaken overnight at 37 ° C; the OD600nm value of the test solution was about 3.0 or more.

4) Collect bacteria, 8000 rpm, 10 min; wash the bacteria once with STE, centrifuge, and weigh (the wet bacteria of 1 L bacterial solution is about 6 g).

5) Add solution I to the ratio of 5 mL of solution I per gram of wet fungus, add 6 mL of 6 g, mix well, and ice bath for 3-5 min.

6) Add freshly prepared solutionII60mL, cover the tube cover, mix it upside down 5-7 times, and leave it at room temperature for 5-7min. The time should not be too long.

7) Add pre-cooled solutionIII45mL (solutionI:solutionII:solutionIII ratio 1:2:1.5), mix upside down 10-20 times, ice bath 10-30min, centrifuge, 11000rpm, 30 Min, the supernatant was removed and centrifuged again for 10 min.

8) Take the supernatant and add 5% CTAB to the final concentration of CTAB 0.18%, while shaking and adding, room temperature for 15 min; room temperature, centrifugation, 11000 rpm, 10 min.

9) The supernatant was decanted, and the precipitate was dissolved with 3 mol/L KAc, about 10 mL, centrifuged, 15 ° C, 11000 rpm, 10 min,

10) remove the supernatant, centrifuge the supernatant, add 0.7 volume of isopropanol, ie 7mL, -20 ° C, precipitation for 30min; 4 ° C, centrifugation 20min, 11000rpm;

11) Discard the supernatant, wash twice with ice-cold 75% ethanol, 11000 rpm, 4 ° C, 5 min; discard the supernatant, precipitate in a 37 ° C incubator, add 10 mL of sterile water to dissolve the precipitate.

12) Detoxification: Add 100 μL of Triton X-114 to 10 mL of the plasmid and mix by shaking. After ice bath for 10 min, then water bath at 42 ° C for 10 min, after turbid state, centrifuge at 11,000 rpm for 10 min at room temperature, carefully take the supernatant liquid in a clean centrifuge tube, and repeat this step until no turbidity occurs in the 42 ° C water bath.

13) Dialysis: The supernatant obtained above was placed in a dialysis bag, and the external permeate was 1 L of distilled water, dialyzed for 24 hours in a 4 ° C chromatography cabinet, and changed every 6 hours. After the dialysis was completed, the solution was dispensed into a 1.5 mL EP tube, 500 μL per tube.

14) Coprecipitation of sodium acetate: An equal volume (i.e., 500 μL per tube) of phenol chloroform (the ratio of phenol to chloroform was 1:1) was added to the above solution, and the EP tube was inverted upside down several times, and centrifuged at room temperature for 11,000 rpm for 10 minutes. The supernatant was taken out in a clean EP tube, 0.7 volume of isopropanol and 1/10 volume of NaAc (3 mol/L, pH 5.2) were added, and the DNA was precipitated at -20 ° C, and centrifuged at 11,000 rpm for 10 min at 30 ° C for 30 min. . Discard the supernatant, wash twice with ice-cold 75% ethanol, 11000 rpm, 4 ° C, 5 min; discard the supernatant, dry the precipitate in an oven at 37 ° C, then add 10 mL of sterilized water to dissolve the precipitate, through a micro spectrophotometer The obtained plasmid was purified and the plasmid concentration was between 700 ng/μL and 1000 ng/μL, and the OD260nm/OD280nm value was between 1.83-1.87. Store at -20 °C for later use.

Construction of lung metastasis model of B and B16 melanoma cells in mice

B16 melanoma cells in logarithmic growth phase, after 0.25% trypsin, the cells were resuspended in medium, washed once with sterile saline, centrifuged, suspended with appropriate amount of physiological saline, and counted with a counting plate. Adjust the cell density to 5 × 10 ⁶ /mL for use. The tail skin of the mice was sterilized with 75% ethanol, and the above B16 cell suspension was inoculated into the tail vein of KM mice, each inoculated with 0.2 mL. That is, each mouse was inoculated with 1 × 10 ⁶ B16 cells. On the 18th day after inoculation, one mouse was randomly sacrificed by spine rupture to determine whether it formed a tumor model.

C, atomization gene therapy

Dispose of PEI stock solution: Weigh 0.215g into a clean glass beaker, add 40mL PBS (pH7.0) and stir with a magnetic stirrer until PEI is completely dissolved, adjust the pH to 7.0 with HCl, and then make up to 50mL with pH7.0PBS. . That is, a PEI solution having a concentration of 4.3 mg/mL (0.1 mol/L N) was prepared. Filter and sterilize with a filter and store at 4 ° C for later use.

Formation of PEI-DNA complex: PEI and DNA were mixed at a ratio of N:P of 10:1 (ie mass ratio of 1.29:1): the plasmid pmTERTp-hmel was adjusted to a concentration of 0.4 mg/mL with ddH ₂ O before the experiment. 5 mL of plasmid was required for each treatment; the concentration of PEI stock solution was adjusted to 0.516 mg/mL to 5 mL with ddH ₂ O; 5 mL of the plasmid was dropped into PEI, mixed, and allowed to stand at room temperature for 30 minutes.

Connect the atomization device (Figure 4), and place 5 B16 melanoma lung metastasis model mice into the glass container of the atomizer. Pour the prepared PEI-DNA complex 10 mL into the nebulizer device. The atomizer was turned on, and compressed air containing 5% CO ₂ was introduced to adjust the gas flow rate to 10 L/min. The PEI-DNA complex dispersed in a mist is introduced into a glass container, and the mouse can breathe freely in the container. Control mice received only an equal amount of PEI. Each group of mice was allowed to breathe for half an hour. Two treatments a week for a total of two weeks. The lungs were dissected 48 hours after the end of the fourth atomization treatment, and the lung formation of each group of mice was as shown in Fig. 5. In the control group, the lungs of No. 1 mice were almost covered by tumor nodules, and the lungs began to smash; the lungs of mice No. 2 and No. 3 were intact, but the tumor nodules were more serious; the number of lung nodules in No. 5 mice was small. However, the diameter was larger and the edge of the lung began to smash; the lungs of the 4th mouse were the best in the control group, but there were also obvious nodules. The lung nodules of the mice in the treatment group almost disappeared.

D, lung histopathology test

The lung tissues of the control and treatment groups were made into paraffin sections and subjected to HE staining. Observe under an inverted microscope and film. As shown in Figure 6, after HE staining of the lung tissue of the control mice, many lung metastases of B16 cells were observed. The tumor cells were round, elliptical or irregular, and the nuclei were deeply stained. Large, obvious shape. In the treatment group, after nebulized gene therapy, HE staining showed that the lung tissue structure was loose, and no nest-shaped metastases were observed.

While the invention has been described with respect to the preferred embodiments of the embodiments of the embodiments of the invention modify. Accordingly, the scope of the invention is defined by the appended claims.

Claims

A targeted anti-cancer gene-plasmid, characterized in that the gene-plasmid is a plasmid containing a tumor-specific promoter and carries a tumor therapeutic gene selected from the group consisting of a tumor suppressor gene and a cytokine gene. And one or both of a pro-apoptotic gene, a toxin protein gene, an angiogenesis gene and a suicide gene, and the plasmid is an eukaryotic expression plasmid.
The gene-plasmid according to claim 1, wherein the tumor-specific promoter is a telomerase reverse transcriptase promoter, an alpha-fetoprotein promoter, a carcinoembryonic antigen promoter, and a prostate-specific antigen promoter. And one of the tissue-specific promoters of breast cancer.
The gene-plasmid according to claim 1, wherein said tumor-bearing therapeutic gene carries two different tumor therapeutic genes simultaneously; or carries a tumor therapeutic gene and a tumor-treating gene expression The product is subjected to a modified protease gene.
The gene-plasmid of claim 3, wherein the tumor therapeutic genes are linked between the tumor therapeutic gene and the protease gene at a ribosome entry site.
The gene-plasmid according to claim 1, wherein the tumor suppressor gene is p53, PTEN, Rb, NF1, VHL or APC; and the cytokine gene is IL-2, IL-12, IL-24. , GM-CSF, INF-α, INF-β, INF-γ; the pro-apoptotic gene is TRAIL, Smac, Omi, Bax, Caspase-3, Caspase-7 or Eorf4; the toxin protein gene is a bee Toxin peptide melittin, scorpion BMK, ribosome inactivating protein gelonin, conotoxin CTX; the suicide gene is cd or tk; the angiogenesis inhibitor gene is endostatin, angiostatin k1-4, blood vessel Produce statin k1-3, plasminogen k5 or sflt-1.
The gene-plasmid of claim 3, wherein the protease gene is Enterokinase, Thrombin, Factor Xa, TEV protease or HRV 3C Protease.
A method for constructing an anti-cancer gene-plasmid, comprising the steps of: a tumor-specific promoter, two tumor therapeutic genes or a tumor therapeutic gene, and a process for modifying a tumor therapeutic gene expression product. Protease gene was cloned into eukaryotic expression In the granule, the two genes are linked by a ribosome entry site; the tumor therapeutic gene is selected from the group consisting of a tumor suppressor gene, a cytokine gene, a pro-apoptotic gene, a toxin protein gene, an angiogenesis gene and a suicide gene.
The method for constructing a targeted anti-cancer gene-plasmid according to claim 7, wherein the tumor suppressor gene is p53, PTEN, Rb, NF1, VHL or APC; and the cytokine gene is IL-2, IL. -12, IL-24, GM-CSF, INF-α, INF-β, INF-γ; the pro-apoptotic gene is TRAIL, Smac, Omi, Bax, Caspase-3, Caspase-7 or Eorf4; The toxin protein gene is melittin melittin, scorpion BMK, ribosome inactivating protein gelonin, conotoxin CTX; the suicide gene is cd or tk; the angiogenesis inhibitor gene is endostatin, angiogenesis Or k1-4, angiostatin k1-3, plasminogen k5 or sflt-1.
The method for constructing a targeted anti-cancer gene-plasmid according to claim 7, wherein the tumor-specific promoter is a telomerase reverse transcriptase promoter, an alpha-fetoprotein promoter, a carcinoembryonic antigen promoter, and prostate specificity. One of a sex antigen promoter and a breast cancer tissue-specific promoter.
Use of a targeted anti-cancer gene-plasmid according to at least one of claims 1 to 6 for the preparation of an antitumor drug;

The targeted anti-oncogene-plasmid and other compounds constitute a pharmaceutical composition selected from the group consisting of a chemotherapeutic drug, a biotoxin, an immunosuppressive compound, and a monoclonal antibody.