Ligand polypeptide and drug delivery system specifically binding to mdscs

Abstract

The invention discloses a ligand polypeptide specifically combined with MDSCs (Myeloid-Derived Suppressor Cells) and a drug delivery system. The ligand polypeptide specifically combined with MDSCs comprises an amino acid sequence as shown in SEQ ID NO.1. The drug delivery system disclosed by the invention comprises the ligand polypeptide, a drug carrying system and at least one active or developing substance, wherein the drug carrying system is specifically lipidosome; the at least one active substance is any substance which is needed to be delivered to a special in-vivo position. The ligand polypeptide disclosed by the invention has good MDSCs-targeting effect, and can be combined with lipidosome to be used as a targeting drug carrier, so that a targeting drug delivery system for tumors is developed.

Description

technical field

[0001] The invention relates to the technical field of protein polypeptides, in particular to a ligand polypeptide specifically combined with MDSCs and a drug delivery system. Background technique

[0002] Tumor is one of the main causes affecting human health. Theoretically speaking, the immune system can recognize the tumor as the non-self of the body, thereby inhibiting the occurrence and development of the tumor, but in fact, the tumor host usually has low immunity and cannot produce effective anti-tumor immunity. In recent years, the tumor immunoediting hypothesis about the interaction between tumor development and the body's immune system has attracted people's attention. The hypothesis holds that tumor cells form a stable tumor immune tolerance pattern by reducing their own immunogenicity, down-regulating the expression of co-stimulatory molecules on the cell surface, releasing a large number of immunosuppressive factors, and recruiting a variety of i...

Examples

Embodiment 1

[0046] Example 1. In vivo phage screening technology to screen MDSCs targeting ligand candidate polypeptides

[0047] The in vivo phage screening technique was used to perform three rounds of screening to obtain the MDSCs targeting polypeptide DPPWLSW (abbreviated as F7, as shown in SEQ ID NO.1). The specific method is as follows:

[0048] (1) In vivo phage screening

[0049] First round of screening:

[0050] 1. Take one tumor-bearing mouse and inject 100 μl Ph.D.-7 into the tail vein TM Phage 7 peptide library (4*10 11 pfu). After 1 hour, 200 μl of 4% chloral hydrate was anesthetized, and 40 ml of PBS was perfused into the myocardium, and then the tumor tissue was collected.

[0051] 2. Cleavage of MDSCs cells: add 100 μl of 1% NP-40 cell lysate to the sorted MDSCs cells, mix well, and lyse on ice for 5 minutes.

[0052] 3. A single colony of ER2738 was inoculated into LB-Tet (LB-tetracycline) medium for overnight culture, and the culture was diluted 1:100 into one 25...

Embodiment 2

[0083] Embodiment 2, have the synthetic material of MDSCs targeting

[0084] This embodiment relates to a synthetic material with MDSCs targeting, which is obtained by linking a derivative polypeptide of MDSC molecule-specific targeting polypeptide with Mal-PEG2000-DSPE. When the sequence of the MDSCs molecule-specific targeting polypeptide is synthesized, GGGC is added to the C-terminus to obtain a derivative polypeptide whose sequence is DPPWLSWGGGC (as shown in SEQ ID NO.3); the free sulfhydryl group on the cysteine ​​of the derivative polypeptide To react with the maleimide group and connect to DSPE-2000-Maleimide (distearoylglyceroylphosphatidylethanolamine polyethylene glycol 2000 maleimide, the polyethylene glycol can also choose PEG5000), A synthetic material with MDSCs targeting is obtained; further, the MDSCs-specific targeting polypeptide can also be directly linked with DSPE-2000-Maleimide; another synthetic material with MDSCs targeting is obtained.

[0085] Th...

Embodiment 3

[0090] Example 3, Derivative polypeptides of MDSCs targeting polypeptides

[0091] 1. Binding experiment of MDSCs, Jurkat and polypeptide DPPWLSW-FITC (suspension cells)

[0092] 1) MDSCs, Jurkat cell counting, 1640 culture medium (10% fetal calf serum) to adjust the cell concentration to 2 × 10 6 pieces / ml. 24-well plate, 200 μl cell suspension per well.

[0093] 2) Under the dark condition, 20 μl of polypeptide DPPWLSW-FITC solution with different concentrations were added to each well, and the concentrations were respectively 1 mM, 500 μM, 50 μM, 5 μM and 0.5 μM. After mixing, 37°C, 5% CO 2 Incubate for 1 h in the dark.

[0094] 3) After the incubation, discard the cell culture medium containing the fluorescent polypeptide, add PBS+2% FBS buffer solution 400 μl / well, 300g, 10min, centrifuge and wash 3 times, thoroughly wash to remove free or non-specifically bound fluorescent polypeptide.

[0095] 4) Using a flow cytometer, count 10,000 cells under an excitation ligh...

technical field

[0001] The invention relates to the technical field of protein polypeptides, in particular to a ligand polypeptide specifically combined with MDSCs and a drug delivery system. Background technique

[0002] Tumor is one of the main causes affecting human health. Theoretically speaking, the immune system can recognize the tumor as the non-self of the body, thereby inhibiting the occurrence and development of the tumor, but in fact, the tumor host usually has low immunity and cannot produce effective anti-tumor immunity. In recent years, the tumor immunoediting hypothesis about the interaction between tumor development and the body's immune system has attracted people's attention. The hypothesis holds that tumor cells form a stable tumor immune tolerance pattern by reducing their own immunogenicity, down-regulating the expression of co-stimulatory molecules on the cell surface, releasing a large number of immunosuppressive factors, and recruiting a variety of i...

Examples

Embodiment 1

[0046] Example 1. In vivo phage screening technology to screen MDSCs targeting ligand candidate polypeptides

[0047] The in vivo phage screening technique was used to perform three rounds of screening to obtain the MDSCs targeting polypeptide DPPWLSW (abbreviated as F7, as shown in SEQ ID NO.1). The specific method is as follows:

[0048] (1) In vivo phage screening

[0049] First round of screening:

[0050] 1. Take one tumor-bearing mouse and inject 100 μl Ph.D.-7 into the tail vein TM Phage 7 peptide library (4*10 11 pfu). After 1 hour, 200 μl of 4% chloral hydrate was anesthetized, and 40 ml of PBS was perfused into the myocardium, and then the tumor tissue was collected.

[0051] 2. Cleavage of MDSCs cells: add 100 μl of 1% NP-40 cell lysate to the sorted MDSCs cells, mix well, and lyse on ice for 5 minutes.

[0052] 3. A single colony of ER2738 was inoculated into LB-Tet (LB-tetracycline) medium for overnight culture, and the culture was diluted 1:100 into one 25...

Embodiment 2

[0083] Embodiment 2, have the synthetic material of MDSCs targeting

[0084] This embodiment relates to a synthetic material with MDSCs targeting, which is obtained by linking a derivative polypeptide of MDSC molecule-specific targeting polypeptide with Mal-PEG2000-DSPE. When the sequence of the MDSCs molecule-specific targeting polypeptide is synthesized, GGGC is added to the C-terminus to obtain a derivative polypeptide whose sequence is DPPWLSWGGGC (as shown in SEQ ID NO.3); the free sulfhydryl group on the cysteine ​​of the derivative polypeptide To react with the maleimide group and connect to DSPE-2000-Maleimide (distearoylglyceroylphosphatidylethanolamine polyethylene glycol 2000 maleimide, the polyethylene glycol can also choose PEG5000), A synthetic material with MDSCs targeting is obtained; further, the MDSCs-specific targeting polypeptide can also be directly linked with DSPE-2000-Maleimide; another synthetic material with MDSCs targeting is obtained.

[0085] Th...

Embodiment 3

[0090] Example 3, Derivative polypeptides of MDSCs targeting polypeptides

[0091] 1. Binding experiment of MDSCs, Jurkat and polypeptide DPPWLSW-FITC (suspension cells)

[0092] 1) MDSCs, Jurkat cell counting, 1640 culture medium (10% fetal calf serum) to adjust the cell concentration to 2 × 10 6 pieces / ml. 24-well plate, 200 μl cell suspension per well.

[0093] 2) Under the dark condition, 20 μl of polypeptide DPPWLSW-FITC solution with different concentrations were added to each well, and the concentrations were respectively 1 mM, 500 μM, 50 μM, 5 μM and 0.5 μM. After mixing, 37°C, 5% CO 2 Incubate for 1 h in the dark.

[0094] 3) After the incubation, discard the cell culture medium containing the fluorescent polypeptide, add PBS+2% FBS buffer solution 400 μl / well, 300g, 10min, centrifuge and wash 3 times, thoroughly wash to remove free or non-specifically bound fluorescent polypeptide.

[0095] 4) Using a flow cytometer, count 10,000 cells under an excitation ligh...

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