CN111592580A - Polypeptide with immune checkpoint CTLA-4 inhibitory activity and application thereof - Google Patents

Abstract

The invention discloses a polypeptide with immune checkpoint CTLA-4 inhibitory activity and application thereof, belonging to the field of biological medicine. The polypeptide of the invention has an amino acid sequence KX_aVLTX_bMSGD (wherein X_a、X_bMay be deleted or substituted) or pharmaceutically acceptable salt thereof, and further, the polypeptide sequence has the function of targeting CTLA-4 after one or more amino acids are deleted, substituted or added on the basis of the sequence, or pharmaceutically acceptable salt of the polypeptide. The polypeptide can specifically recognize CTLA-4 on the surface of activated T cells and relieve immunosuppression caused by CTLA-4/B7 signal pathways, thereby achieving the purpose of killing tumor cells.

Description

Polypeptide with immune checkpoint CTLA-4 inhibitory activity and application thereof

Technical Field

The invention belongs to the field of biological medicines, and relates to a polypeptide with immune checkpoint inhibitory activity and application thereof.

Background

Immune checkpoints are a series of immunosuppressive molecules, a variety of molecules including Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). Under normal physiological conditions, immune checkpoints can modulate immune responses, prevent self-tissue damage, and have important implications for maintaining immune tolerance, while immune checkpoints are associated with many autoimmune diseases, such as lupus-like autoimmune syndrome and autoimmune dilated cardiomyopathy. However, during the development of tumor, the activation and high expression of immune checkpoint molecules can inhibit the function of immune cells, weaken the killing capability of the immune cells to tumor cells and mediate the immune escape of the tumor. Immune checkpoint blockers are therapeutic approaches that enhance anti-tumor immune responses by inhibiting negative regulatory signals, modulating immune cell activity.

Throughout the course of the immune response, dual signaling pathways are required for T cell activation. Firstly, recognizing antigen presenting cell APC or MHC molecule antigen peptide complex on the surface of tumor cell through T cell receptor TCR specificity on the surface of T cell; second, co-stimulatory molecules on the APC or tumor cell bind to co-stimulatory receptors on the T cell, activating or inhibiting the T lymphocyte. Costimulatory molecules on T cells mainly include the coactivator 4-1BB, CD27, CD28, OX40, ICOS, and the coactivator CTLA4, PD-1.

Wherein the CTLA-4 is also named as CD152, and is a compoundLeukocyte differentiation antigensIt is a transmembrane protein expressed by activated effector T cells, which inhibits the proliferation of T cells and also inhibits the relevant cell cycle processes and the secretion of cytokines such as interleukin-2 (IL-2), interferon gamma (IFN-gamma) to varying degrees. CTLA-4 exerts its inhibitory function through a variety of mechanisms, including competitive binding to co-stimulatory signal CD28 to a common ligand B7(CD80/CD86) on Antigen Presenting Cells (APCs). Antibodies targeting CTLA-4 are currently used to treat a variety of human malignancies with the aim of blocking CTLA-4 inhibition during T cell activation.

As CTLA-4 has been studied more and more deeply, it has been found to play a role also on regulatory T cells (Tregs) in the tumor microenvironment. CTLA-4 is highly expressed on regulatory T cells (Tregs) in a tumor microenvironment, and antibodies targeting CTLA-4 may eliminate Treg cells in the tumor microenvironment by mediating Antibody dependent phagocytosis (ADCP) of macrophages or Antibody Dependent Cellular Cytotoxicity (ADCC) of natural killer cells, thereby achieving an anti-tumor effect.

Iplilimumab is a monoclonal antibody (mAb) against CTLA-4 immunoregulation, the single drug indication is melanoma, and with the rise of combination, the tumor spectrum of CTLA-4 targeted drugs involved in treatment is expanding, for example, it has been shown to have significant effect on melanoma, lung cancer, and renal cancer in combination with PD-1 antibody. In addition, the combination of CTLA-4 target in Chimeric Antigen Receptor T, CAT-T therapy is a major focus of immunotherapy research.

Disclosure of Invention

1. Problems to be solved

The polypeptide acts on CTLA-4 to inhibit a CTLA-4/B7 pathway, and a plurality of experimental models verify that the sequence has good tumor inhibition effect and has great development prospect.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

a polypeptide having immune checkpoint CTLA-4 inhibitory activity, wherein: has KXaVLTXBMSGD or a pharmaceutically acceptable salt thereof, wherein Xa and Xb may be deleted or substituted by an amino acid.

The polypeptide, characterized in that: the polypeptide sequence is a polypeptide which still has the function of targeting CTLA-4 after one or more amino acids are deleted, substituted or added on the basis of the sequence of claim 1, or a pharmaceutically acceptable salt of the polypeptide.

The polypeptide, characterized in that the polypeptide sequence is KMVLTMMSGD.

The application of the polypeptide in preparing the medicine for preventing and treating the tumor.

Specifically, the method comprises the following steps:

1. method for solid phase synthesis of protein with KX_aVLTX_bMSGD (wherein X_a、X_bCan be deleted or replaced), and the purity and molecular weight of the polypeptide are detected by HPLC and mass spectrometry, taking the sequence KMVLTMMSGD as an example, and the detailed process is shown in example 1;

2. connecting FITC to a polypeptide chain to enable the polypeptide chain to carry a fluorescent label, and detecting the combination condition of the fluorescent-labeled polypeptide and T cells at different concentrations by using a flow cytometer, taking a sequence KMVLTMMSGD as an example, wherein the detailed process is shown in an example 2-3;

negative regulatory function of CTLA-4 inhibits T cell activation, adding the sequence KX_aVLTX_bMSGD (wherein X_a、X_bCan be deleted or replaced) can enhance the activation degree of the immune system and improve the secretion quantity of IL-2, taking the sequence KMVLTMMSGD as an example, the detailed process is shown in example 4;

4. detecting the in vitro activity of the polypeptide, incubating immune cells and tumor cells, and adding CTLA-4 antagonistic peptide KX_aVLTX_bMSGD (wherein X_a、X_bCan be deleted or replaced), can enhance the killing capability of the immune system to tumor cells, and respectively detects the killing capability to melanoma, non-small cell lung cancer, renal cell carcinoma, ovarian cancer, head and neck cancer, prostatic cancer, breast cancer and colon cancer, taking sequence KMVLTMMSGD as an example, the detailed process is shown in example 5;

5. detecting in vivo anti-tumor activity of polypeptide, establishing subcutaneous transplantation tumor model, and administering CTLA-4 antagonistic peptide KX in the experimental process_aVLTX_bMSGD (wherein X_a、X_bCan be deleted or replaced), can inhibit tumor growth, and can evaluate the in vivo efficacy of the polypeptide by respectively detecting the tumor inhibition rate of the polypeptide on different tumors including melanoma, non-small cell lung cancer, renal cell carcinoma, ovarian cancer, head and neck cancer, prostate cancer, breast cancer and colon cancer, taking sequence KMVLTMMSGD as an example, the detailed process is shown in example 6;

6. and (3) detecting the safety of the polypeptide, using the caenorhabditis elegans as a carrier, evaluating the influence of the polypeptide on the larval mortality and the head swing frequency of the nematode, taking the sequence KMVLTMMSGD as an example, and evaluating the safety of the polypeptide, wherein the detailed process is shown in examples 7-8.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) the polypeptide of the invention has simple structure, is easy to synthesize, separate and purify, effectively inhibits a CTLA-4/B7 path, and eliminates the effect of immunosuppression;

(2) the adverse reaction and toxic and side reaction of the polypeptide are weak;

(3) the polypeptide has good effect of eliminating immunosuppression in vivo and in vitro models, and has the specific effects of obviously improving the activity of T cells and strengthening the killing effect on tumor cells so as to generate tumor inhibition effect.

Drawings

FIG. 1 HPLC purity analysis of the polypeptide.

FIG. 2 polypeptide MS detection map.

FIG. 3 flow cytometry detects binding of T cells to FITC-anti-CTLA-4 polypeptides. A: the binding rate of T cells to 1. mu.M polypeptide; b: the binding rate of T cells to 5. mu.M polypeptide; c: t cells bind to 10. mu.M polypeptide.

FIG. 4 confocal laser microscopy of T cell binding to FITC-anti-CTLA-4 polypeptide. A: DiI-cell membrane B: FITC-anti-CTLA-4 polypeptide C: merge graph.

FIG. 5 Effect of the polypeptide on IL-2 in supernatants of SEB-stimulated PBMC experiments. Note: p <0.05, P <0.01, P <0.001, ns indicates P >0.05, compared to the control group (SEBalone group) and to the Ipilimumab group.

FIG. 6 Effect of the polypeptide on the stimulation of IFN-. gamma.in the supernatant of PBMC by SEB. Note: p <0.05, P <0.01, P <0.001, ns indicates P >0.05, compared to the control group (SEBalone group) and to the Ipilimumab group.

Detailed Description

The invention is further described with reference to specific examples. The following description is only exemplary of the present invention, and is not intended to limit the present invention in any way, and those skilled in the art can modify the present invention by applying the above-described technical disclosure to equivalent embodiments with equivalent modifications. Without departing from the scope of the invention, any simple modification or equivalent changes made to the following examples, given as sequence KMVLTMMSGD in the following embodiments, according to the technical essence of the invention, fall within the scope of the invention:

example 1

Preparation of targeted CTLA-4 polypeptides

The anti-CTLA-4 polypeptides may be prepared by methods well known in the art, for example, by solid phase synthesis of polypeptides, or by a polypeptide synthesizer. The invention adopts Fomc solid phase polypeptide synthesis method to synthesize anti-CTLA-4 polypeptide, the specific synthesis steps are as follows:

(1) swelling the resin, adding the first amino acid;

(2) adding a second amino acid and subsequent peptide chain extension;

(3) polypeptide cleavage;

(4) HPLC analysis of the polypeptide and purification of the polypeptide;

(5) and (4) mass spectrum identification.

The polypeptide is synthesized by adopting the standard Fmoc scheme, which is characterized in that the first Fmoc-amino acid carboxyl at the C terminal of the peptide to be synthesized is connected with resin in a covalent form, and then the N segment of the amino acid is used as the starting point of the polypeptide synthesis, and the peptide bond is changed into the peptide bond in the next year by carrying out dehydration condensation reaction with the carboxyl terminal of the next amino acid. And (3) deprotecting the protection of Fmoc-amino acid at the N terminal, reacting the N terminal of the second amino acid with the carboxyl terminal of the following amino acid, and repeating the steps until the polypeptide synthesis is finished. In the process, after the resin is condensed with the first amino acid with a protecting group of the target polypeptide, the protecting agent is removed, then the second amino acid is added for condensation, the last amino acid is guided to be added by repeating the steps, after the protecting agent is removed, the polypeptide is cut off from the resin by utilizing a cutting reagent, ether precipitation is carried out, and the peptide is obtained after washing. And (3) carrying out mass spectrometry and purification on the synthesized polypeptide, wherein the purity of the synthesized polypeptide is more than 95%, and thus obtaining the CTLA-4 synthetic peptide. ESI-MS mass spectrometry analysis and determining that the molecular weight of the synthetic peptide is consistent with the theoretical molecular weight, and freezing and storing at-20 ℃ for later use. The HPLC chart is shown in FIG. 1, and the molecular weight detection chart is shown in FIG. 2.

Example 2

Detection of specific binding of a polypeptide to a target at the cellular level.

The flow cytometry is a technology for exciting a single unidirectional flowing particle by a laser beam, detecting scattered light of the particle and a fluorescent marker carried by the particle, and thus rapidly detecting and analyzing a plurality of physical characteristics of the single particle and sorting cells. Is widely applied to scientific research and clinical medical examination, and is the most advanced cell quantitative analysis technology in the present generation. The invention relates to a polypeptide with cancer prevention and/or tumor inhibition activity, which has a main target point of CTLA-4, wherein the designed anti-CTLA-4 polypeptide is connected with an FITC fluorescent marker, and the binding capacity of the polypeptide and cell surface C TLA-4 on the cell level is reflected by the binding rate of cells and an FITC-anti-CTLA-4 polypeptide solution.

1 materials of the experiment

T cells are obtained by extraction and purification, BSA confining liquid, Ficoll reagent, sorting magnetic beads and the like.

2 laboratory instruments

CO₂Cell culture case, flow cytometer, magnetic bead sorting frame, magnetic bead sorting column, horizontal centrifuge, liquid nitrogen tank, super clean bench, small centrifuge, electronic balance, full-automatic high-pressure steam sterilization pot, oven.

3, experimental method:

t cells containing 1640 medium were collected and washed twice with ice-cold PBS. 1ml of a 1% BSA solution was added, fixed on a rotary mixer, and mixed at 4 ℃ for 30 min. Add 10. mu.l of 1mg/ml FITC-anti-CTLA-4 polypeptide solution in dark place, fix it on a rotary mixer, and mix it at 4 ℃ for 1h in dark place. After the incubation, the supernatant was discarded after centrifugation at 800rpm for 5 min. Wash once with ice-cold PBS. The cell concentration was adjusted with PBS to 1X 106 cells/ml. 3 replicates per cell were prepared, 0.5ml per sample.

And detecting the combination of the polypeptide and the CTLA-4 by using a flow cytometer, and sequentially turning on a stabilized voltage power supply, a transformer, a flow cytometer host, a computer and a printer from left to right. The flow drawer was opened and purified water was added to the sheath fluid bucket until 2/3 was reached. The waste solution was decanted and 200ml of sodium hypochlorite solution containing 10% available chlorine was added. The hydraulic valve is adjusted to the pressure position to remove air bubbles between the flow line and the filter. The sample tube was removed and PRIME function was performed twice, 1ml PBS, HINGRUN 2 min. The measurement is started and the sample is analyzed. After the measurement is completed, the sample holder is moved to the left and 1ml of FACSClean is evacuated. The sample holder was then returned to the positive position for HING RUN 5 min. FACS Clean was changed to purified water, the sample holder was moved to the left, 1ml was extracted under vacuum, the sample holder was returned to the right, HING RUN 10 min. The sample tube was removed and PRIME function was performed twice per Standby. Finally, 1ml of purified water is left in the flow test tube. The cell was operated in Stan dby for 20min, after fan cooling the laser, the flow cytometer was turned off. And exiting the program and closing the computer.

4 results of the experiment

Binding of T cells to FITC-conjugated anti-CTLA-4 polypeptide is shown in FIG. 3, where A, B, C is the binding rate of T cells to FITC-conjugated anti-PD-1 polypeptide at 1. mu.M, 5. mu.M, and 10. mu.M. 14.8%, 29.6% and 49.9%, respectively, demonstrated that the polypeptide could bind to CTLA-4 expressing T cells.

Example 3

Qualitative detection experiment-fluorescence imaging experiment for specific binding of polypeptide to target at cellular level

1 materials of the experiment

T cells obtained by extraction and purification. The culture conditions are as follows: the 1640 culture medium containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) was used at 37 ℃ and 5% CO₂The culture box is cultured to logarithmic growth phase.

2 laboratory instruments

CO₂Cell culture case, single channel pipettor, fluorescence microscope, inverted microscope, liquid nitrogen tank, superclean bench, small centrifuge, electronic balance.

3 Experimental methods

Adjusting T cell concentration to 1 × 10⁵cells/ml in laser copolymerizationOn the coke dish. Washed twice with ice-cold PBS. 1ml of a 1% BSA solution was added thereto, and mixed at 4 ℃ for 30 min. Add 10. mu.L of 1mg/ml FITC-anti-CTLA-4 polypeptide solution in dark and mix at 4 ℃ for 1 h. After incubation with the drug was complete, one wash with ice-cold PBS. DiI dye was added and washed twice with ice-cold PBS.

Detecting the combination of the polypeptide and the CTLA-4 by using a laser confocal microscope, fixing parameters, respectively shooting in proper channels, superposing fluorescent images of different channels, and judging the combination of the polypeptide and the CTLA-4.

4 results of the experiment

The results are shown in FIG. 4, which shows a significant yellowing after Merge, and again verifies that the anti-CTLA-4 polypeptide can be combined with T cells and combined on the surface of cell membranes.

Example 4

And detecting the influence of the anti-CTLA-4 polypeptide on the stimulation of the cell factors in the PBMC system by the SEB.

SEB (Staphyloccalen tetroxins), staphylococcal enterotoxin B, is a superantigen and a very effective T cell activator, can activate immune cells at a very low concentration, and can interact with CD28, MHC class II molecules and TCR, so that a stable synaptic structure is formed between an antigen presenting cell and the T cell, and the T cell is effectively activated. In the process, CTLA-4 antagonistic peptide is added to effectively enhance the activation of T cells, and the activation condition of the T cells is evaluated by detecting the contents of cell factors IL-2 and IFN-gamma in supernatant.

1 materials of the experiment

The kit comprises human peripheral blood of healthy volunteers, Staphylococcal Enterotoxin B (SEB), human IL-2 cytokines, a human IL-2 detection kit and a human IFN-gamma detection kit.

2 laboratory instruments

Horizontal centrifuge, cell counter, electronic balance (d ═ 0.01mg), microplate reader, and super clean bench.

3 Experimental methods

Using SEB with the concentration of 2.5ng/mL⁺1640 culture Medium for adjusting PBMC concentration to 10⁶one/mL, added to different areas of a 96-well plate at 100. mu.L/wellGood mark, 10 per well⁵And (5) PBMCs. Because the drug and cells were added at a ratio of 1:1, SEB was used at a concentration of 2.5ng/mL⁺1640 medium to 40. mu.M, and then to 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M for filter sterilization. After administration at 37 ℃ CO₂Culturing in incubator for 72h, taking out 96-well plate, centrifuging for 10min at 300 × g, 3 g, 10min in horizontal centrifuge, collecting supernatant, storing at-20 deg.C for inspection, collecting supernatant, thawing at room temperature, preparing standard curve according to steps in human cytokine IL-2 and IFN-gamma kit, detecting sample, making three duplicate wells for each group, calculating cytokine level of each group according to standard curve of each cytokine, statistically analyzing with Graph Padprism8.0.2 software, comparing after three times of each experiment value, comparing between groups with T test, analyzing with one-factor ANOVA among groups, and when P is P, analyzing with one-factor ANOVA<Differences were considered statistically significant at 0.05 (. about.P)<0.05，**P<0.01，***P<0.001)。

4 results of the experiment

From figure 5, it can be seen that the CTLA-4 antagonistic peptide can significantly increase the IL-2 content in the supernatant, and the administered group has a significant difference compared to the control group, demonstrating that the polypeptide can significantly enhance the degree of T cell activation in the system. As can be seen from figure 6, the CTLA-4 antagonistic peptide can significantly improve the IFN-gamma content in the supernatant, and the administration group has significant difference compared with the control group, which proves that the polypeptide can significantly enhance the activation degree of T cells in the system.

Example 5

Detecting the in vitro anti-tumor activity of the polypeptide, adding CTLA-4 antagonistic peptide to enhance the killing capacity of an immune system to tumor cells after the immune cells and the tumor cells are incubated together, and respectively detecting the killing capacity to melanoma, non-small cell lung cancer, renal cell carcinoma, ovarian cancer, head and neck cancer, prostate cancer, breast cancer and colon cancer after the polypeptide is added, taking a sequence KMVLTMMSGD as an example, the selected cell line is as follows: human melanoma cells (ME), human non-small cell lung cancer cells (A549), human renal cell carcinoma cells (769-P), ovarian cancer cells (A2780), oral squamous cell carcinoma in head and neck squamous cell carcinoma (SCC-4), prostate cancer (DU-145), breast cancer (BT474), human colon cancer cells (HT-29).

Lactate Dehydrogenase (LDH) is abundant in cytoplasm, when the cell state is normal, the LDH cannot pass through a cell membrane, but when the cell is damaged or dies, the LDH can be released out of the cell, and the content of the LDH in the supernatant is in direct proportion to the death number of the cell, so that the killing capacity of the T cell to the tumor cell after the administration is determined by detecting the content of the LDH in the supernatant of a co-incubation system.

1 materials of the experiment

The eight selected human tumor cell lines, the LDH detection kit, the human peripheral blood lymphocyte separation medium and the IL-2 cell factor.

2 laboratory instruments

Horizontal centrifuge, single channel pipettor, ELIASA, small centrifuge, electronic balance, pH meter, cell counter, and cell culture box.

3 Experimental methods

Selecting eight tumor cell lines, culturing in 1640 culture solution containing 10% Fetal Bovine Serum (FBS), penicillin (100kU/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO₂The culture box is cultured to logarithmic growth phase. The cell concentration was adjusted to the desired concentration and seeded in a 96-well plate. At 37 deg.C, 5% CO₂The incubator of (2) is cultured for 24 hours. Co-incubation reactions were subsequently used to demonstrate the effect of blockade of the CTLA-4 pathway on tumor cells. Peripheral Blood Mononuclear Cells (PBMC) were extracted from blood, while tumor cells were cultured, and then the two cells were co-incubated at a certain ratio to set polypeptide concentrations of 1. mu.M, 5. mu.M, and 10. mu.M, and after administration, they were cultured in a cell culture chamber at 37 ℃ for 2 days. After 2 days, the supernatant was removed and the secretion of LDH was measured using the kit. Statistical analysis was performed using SPSS 19.0(SPSS inc. chicago, IL, USA) software. Results are expressed as mean ± SD. Group comparisons were performed using the T-test, and single-factor ANOVA analysis was used between groups. When P is present<Differences were considered statistically significant at 0.05 (. about.P)<0.05，**P<0.01， ***P<0.001)。

4 results of the experiment

After the polypeptides are added into a co-incubation system, the killing conditions of the T cells on eight tumor cells are shown in tables 1-8.

TABLE 1 polypeptide killing of ME cells in Co-incubation systems

Group of	Polypeptide dose (mu M)	Cytotoxicity (%)
			Control group	/	9.21±0.38
Polypeptide low dose group	1.25	12.22±0.21*
			Polypeptide medium dose group	5	17.48±0.31*
High dose group of polypeptides	10	19.72±0.42*

Note: p <0.05 compared to control.

Changes in LDH in the co-incubation system reflect the effect of the polypeptide on ME cells in the co-incubation system. Compared with a control group, the administration group has significant difference, which indicates that the polypeptide can enhance the activation degree of an immune system and promote the killing effect of T cells on tumor cells ME.

TABLE 2 polypeptide killing of A549 cells in Co-incubation systems

Group of	Polypeptide dose (mu M)	Cytotoxicity (%)
			Control group	/	10.37±0.42
Polypeptide low dose group	1.25	13.16±0.27*
			Polypeptide medium dose group	5	18.66±0.37*
High dose group of polypeptides	10	19.94±0.22*

Note: p <0.05 compared to control.

Change of LDH in co-incubation system reaction polypeptide effects on a549 cells in co-incubation system. Compared with a control group, the administration group has significant difference, which indicates that the polypeptide can enhance the activation degree of an immune system and promote the killing effect of T cells on tumor cells A549.

TABLE 3 polypeptide versus 769-P cell killing in Co-incubation systems

Group of	Polypeptide dose (mu M)	Cytotoxicity (%)
			Control group	/	9.85±0.29
Polypeptide low dose group	1.25	12.66±0.47*
			Polypeptide medium dose group	5	16.94±0.17*
High dose group of polypeptides	10	18.12±0.35*

Note: p <0.05 compared to control.

Change in LDH in the Co-incubation System the effect of the polypeptide on 769-P cells in the Co-incubation System. Compared with a control group, the significant difference of the administration group and the control group indicates that the polypeptide can enhance the activation degree of an immune system and promote the killing effect of T cells on tumor cells 769-P.

TABLE 4 polypeptide killing of A2780 cells in a co-incubation System

Group of	Polypeptide dose (mu M)	Cytotoxicity (%)
			Control group	/	10.51±0.14
Polypeptide low dose group	1.25	12.26±0.27*
			Polypeptide medium dose group	5	18.75±0.36*
High dose group of polypeptides	10	21.47±0.18*

Note: p <0.05 compared to control.

Changes in LDH in the co-incubation system reflect the effect of the polypeptide on a2780 cells in the co-incubation system. Compared with a control group, the administration group has significant difference, which indicates that the polypeptide can enhance the activation degree of an immune system and promote the killing effect of T cells on tumor cells A2780.

TABLE 5 polypeptide killing of SCC-4 cells in Co-incubation systems

Group of	Polypeptide dose (mu M)	Cytotoxicity (%)
			Control group	/	9.21±0.19
Polypeptide low dose group	1.25	12.27±0.16*
			Polypeptide medium dose group	5	15.39±0.15*
High dose group of polypeptides	10	18.72±0.37*

Note: p <0.05 compared to control.

Change in LDH in co-incubation systems the effect of the polypeptide on SCC-4 cells in co-incubation systems. The significant difference of the administration group and the control group indicates that the polypeptide can enhance the activation degree of an immune system and promote the killing effect of T cells on the SCC-4 of tumor cells.

TABLE 6 killing of DU-145 cells by the polypeptide in Co-incubation systems

Group of	Polypeptide dose (mu M)	Cytotoxicity (%)
			Control group	/	10.15±0.14
Polypeptide low dose group	1.25	16.78±0.36*
			Polypeptide medium dose group	5	18.24±0.16*
High dose group of polypeptides	10	19.62±0.25*

Note: p <0.05 compared to control.

Changes in LDH in the co-incubation systems reacted the effect of the polypeptide on DU-145 cells in the co-incubation systems. Compared with a control group, the significant difference of the administration group and the control group indicates that the polypeptide can enhance the activation degree of an immune system and promote the killing effect of T cells on tumor cells DU-145.

TABLE 7 killing of BT474 cells by the polypeptide in Co-incubation System

Group of	Polypeptide agentsAmount (μ Μ)	Cytotoxicity (%)
			Control group	/	12.32±0.19
Polypeptide low dose group	1.25	14.63±0.25*
			Polypeptide medium dose group	5	18.92±0.16*
High dose group of polypeptides	10	22.56±0.21*

Note: p <0.05 compared to control.

Changes in LDH in co-incubation systems reflect the effect of the polypeptide on BT474 cells in co-incubation systems. The significant difference of the administration group and the control group indicates that the polypeptide can enhance the activation degree of an immune system and promote the killing effect of T cells on tumor cells BT 474.

TABLE 8 polypeptide killing of HT-29 cells in Co-incubation systems

Group of	Polypeptide dose (mu M)	Cytotoxicity (%)
			Control group	/	9.25±0.14
Polypeptide low dose group	1.25	13.62±0.13*
			Polypeptide medium dose group	5	15.36±0.13*
High dose group of polypeptides	10	18.23±0.26*

Note: p <0.05 compared to control.

Changes in LDH in the co-incubation systems reacted to the effect of the polypeptide on HT-29 cells in the co-incubation systems. The significant difference between the administration group and the control group indicates that the polypeptide can enhance the activation degree of an immune system and promote the killing effect of T cells on tumor cells HT-29.

Example 6

The in vivo anti-tumor activity of the anti-CTLA-4 polypeptide is detected, and the in vivo anti-tumor activity of the polypeptide is evaluated by establishing a subcutaneous transplantation tumor model according to the tumor inhibition rates of the polypeptide on human melanoma cells (ME), human non-small cell lung cancer cells (A549), human renal cell carcinoma cells (769-P), ovarian cancer cells (A2780), oral squamous cell carcinoma (SCC-4) in head and neck squamous cell carcinoma, prostate cancer (DU-145), breast cancer (BT474) and human colon cancer cells (HT-29).

1 materials of the experiment

Balb/c nude mice, anti-CTLA-4 polypeptide, lymphocyte separation liquid, the above mentioned eight tumor cells, IL-2 cell factor, 1640 culture medium, electronic vernier caliper.

2 method of experiment

Adult female Balb/c nude mice are purchased and bred in the barrier environment of the pharmaceutical animal experiment center of Chinese pharmaceutical university, the circadian rhythm of the animal house is alternated with constant temperature and humidity for 12h, after the mice are adaptively fed for one week, the nude mice are randomly grouped into 5 mice in each group, namely a PBMC group, a 5mg/kg group, a 10mg/kg group, a 20mg/kg group and an Iplilimumab group, then the tumor cells are digested and collected, PBS is washed for 1 time, physiological saline is resuspended, meanwhile, the activated PBMC is centrifugally collected, PBS is washed for 1 time, the physiological saline is resuspended and then mixed with the tumor cells, so that the concentration of the tumor cells in the mixed suspension is 6 × 10⁶Per mL, PBMC concentration 3 × 10⁶one/mL. The mouse left axilla was sterilized by wiping the skin with 75% alcohol cotton, and then the cell mixed suspension was inoculated subcutaneously with 100. mu.L of each mouse into the left axilla using a 1mL sterile syringe, and after completion of the inoculation, the mice were housed in cages for group rearing, in which the PBMC group, 5mg/kg group, 10mg/kg group, 20mg/kg group, and Iplilimumab group were re-injected with PBMC at the 14 th tail vein after the inoculation to enhance the reconstitution of the immune system of the mice. Mice were weighed every three days and the subcutaneous tumor size, tumor volume V (mm) was measured with a vernier caliper³)＝a×b²/2(a is the length of the subcutaneous tumour and b is the width of the subcutaneous tumour). Tumor cells were inoculated and dosed as per table 9;

note: iplilimumab, marketed under the trade name Yervoy, is a monoclonal antibody that effectively blocks a molecule called cytotoxic T cell antigen-4 (CTLA-4) and is available from Selleckchem.

TABLE 9 polypeptide CT-2 in vivo anti-tumor Activity dosing regimen

The eight tumor cells were tested according to the above procedures, and 28 days later, the mice were weighed, then the mice were sacrificed by cervical dislocation, and the tumor tissue was dissected out and weighed to calculate the tumor inhibition rate. Group comparisons were performed using the T-test, and single-factor ANOVA analysis was used between groups. Differences were considered statistically significant when P <0.05 (. P <0.05,. P <0.01,. P < 0.001). .

3 results of the experiment

The tumor inhibition rates of the polypeptides on eight tumors are shown in tables 10-17.

TABLE 10 tumor inhibition Rate of Polypeptides against melanoma cells (ME)

Group of	Dosage to be administered	Tumor inhibition Rate (%)
			PBMC group	/	9.71±0.46
Polypeptide low dose group	5mg/kg	22.47±2.21
			Polypeptide medium dose group	10mg/kg	32.48±4.31
High dose group of polypeptides	20mg/kg	40.72±3.42*
			Iplilimumab group	100μg/kg	52.37±4.68**

Note: p <0.05, P <0.01, compared to control (PBMC group).

The polypeptide can obviously inhibit the growth of melanoma cells (ME) in a mouse body compared with a control group at a high dose, and has a good tumor inhibition effect.

TABLE 11 tumor inhibition ratio of human non-small cell lung carcinoma cells (A549) by the polypeptide

Group of	Dosage to be administered	Tumor inhibition Rate (%)
			PBMC group	/	10.35±1.46
Polypeptide low dose group	5mg/kg	20.73±2.47
			Polypeptide medium dose group	10mg/kg	34.59±6.18
High dose group of polypeptides	20mg/kg	45.74±2.41*
			Iplilimumab group	100μg/kg	56.95±6.62**

Note: p <0.05, P <0.01, compared to control (PBMC group).

When the polypeptide is in high dose, compared with a control group, the polypeptide can obviously inhibit the growth of human non-small cell lung cancer cells (A549) in mice, and has good tumor inhibition effect.

TABLE 12 tumor inhibition rate of polypeptide on human renal cell carcinoma cell (769-P)

Group of	Dosage to be administered	Tumor inhibition Rate (%)
			PBMC group	/	7.91±1.71
Polypeptide low dose group	5mg/kg	15.92±3.74
			Polypeptide medium dose group	10mg/kg	25.63±2.51
High dose group of polypeptides	20mg/kg	38.14±1.71*
			Iplilimumab group	100μg/kg	49.98±4.46**

Note: p <0.05, P <0.01, compared to control (PBMC group).

When the polypeptide is in high dose, the polypeptide can obviously inhibit the growth of renal cell carcinoma cells (769-P) in a mouse body compared with a control group, and has good tumor inhibition effect.

TABLE 13 tumor inhibition rates of polypeptides on human ovarian cancer cells (A2780)

Group of	Dosage to be administered	Tumor inhibition Rate (%)
			PBMC group	/	9.50±1.34
Polypeptide low dose group	5mg/kg	19.02±2.27
			Polypeptide medium dose group	10mg/kg	31.73±5.67
High dose group of polypeptides	20mg/kg	41.95±2.21*
			Iplilimumab group	100μg/kg	55.82±3.29**

Note: p <0.05, P <0.01, compared to control (PBMC group).

When the polypeptide is in high dose, compared with a control group, the polypeptide can obviously inhibit the growth of human ovarian cancer cells (A2780) in a mouse body, and has good tumor inhibition effect.

TABLE 14 tumor inhibition Rate of Polypeptides against oral squamous cell carcinoma in head and neck (SCC-4)

Group of	Dosage to be administered	Tumor inhibition Rate (%)
			PBMC group	/	11.36±0.44
Polypeptide low dose group	5mg/kg	26.29±2.12
			Polypeptide medium dose group	10mg/kg	38.00±4.14
High dose group of polypeptides	20mg/kg	47.64±3.28*
			Iplilimumab group	100μg/kg	60.44±5.01**

Note: p <0.05, P <0.01, compared to control (PBMC group).

The polypeptide can obviously inhibit the growth of oral squamous cell carcinoma (SCC-4) in mice compared with a control group at high dose, and has good tumor inhibition effect.

TABLE 15 tumor inhibition rate of the polypeptide on prostate cancer (DU-145)

Group of	Dosage to be administered	Tumor inhibition Rate (%)
			PBMC group	/	8.92±1.51
Polypeptide low dose group	5mg/kg	17.87±2.57
			Polypeptide medium dose group	10mg/kg	29.81±6.43
High dose group of polypeptides	20mg/kg	39.41±2.51*
			Iplilimumab group	100μg/kg	50.11±2.14**

Note: p <0.05, P <0.01, compared to control (PBMC group).

When the polypeptide is in high dose, the growth of the prostate cancer (DU-145) in a mouse can be remarkable compared with a control group, and the polypeptide has a good tumor inhibition effect.

TABLE 16 tumor inhibition rate of polypeptide against breast cancer (BT474)

Group of	Dosage to be administered	Tumor inhibition Rate (%)
			PBMC group	/	8.78±1.90
Polypeptide low dose group	5mg/kg	17.67±4.15
			Polypeptide medium dose group	10mg/kg	28.44±2.79
High dose group of polypeptides	20mg/kg	42.34±1.90*
			Iplilimumab group	100μg/kg	55.69±3.31**

Note: p <0.05, P <0.01, compared to control (PBMC group).

When the polypeptide is used at a high dose, compared with a control group, the polypeptide can obviously inhibit the growth of breast cancer (BT474) in a mouse body, and has a good tumor inhibition effect.

TABLE 17 tumor inhibition rates of polypeptides on human colon cancer cells (HT-29)

Group of	Dosage to be administered	Tumor inhibition Rate (%)
			PBMC group	/	7.26±1.57
Polypeptide low dose group	5mg/kg	14.61±3.43
			Polypeptide medium dose group	10mg/kg	23.51±2.30
High dose group of polypeptides	20mg/kg	40.99±1.57*
			Iplilimumab group	100μg/kg	57.71±3.12**

Note: p <0.05, P <0.01, compared to control (PBMC group).

When the polypeptide is used at a high dose, compared with a control group, the polypeptide can obviously inhibit the growth of human colon cancer cells (HT-29) in a mouse body, and has a good tumor inhibition effect.

Example 7

Detection of acute toxicity of anti-CTLA-4 polypeptide to caenorhabditis elegans

1 materials of the experiment

N2 wild type caenorhabditis elegans, CTLA-4 resisting polypeptide, a culture dish, polypeptone, agar powder and sodium hypochlorite.

2 method of experiment

OP50 E.coli was cultured and transferred every other day to 150mL of LB medium for culture. Placing caenorhabditis elegans on OP50 Escherichia coli liquid, controlling room temperature at 20 deg.C for synchronization of caenorhabditis elegans, and allowing larva to stay in L1 stage by cracking into adult and controlling nutrition. The administration group was set to three concentrations of 5. mu.M, 10. mu.M and 20. mu.M, and 50. mu.LOP 50 E.coli strain and 40L 1 larvae (50. mu.L) were added to each well of a 96-well plate. Blank group 100. mu.L/well M9 buffer was added to the experimental group 100. mu.L/well anti-CTLA-4 polypeptide (5. mu.M, 10. mu.M, 20. mu.M) was added according to a dose gradient. Each set of 4 duplicate wells and ensuring a final volume of 200 μ L per well. After the drug adding treatment is finished, the number of nematodes per hole in the initial state is observed and recorded as N₀Then placing the nematodes in an electric heating constant temperature incubator at 20 ℃ for culturing for 24h, and recording the number N of the nematodes in each hole after 24h_t. Statistical analysis was performed using the SPSS17.0 mathematical statistics software package, mean + -SD. The comparison between groups was performed by T test, P<0.05 had a significant statistical difference.

3 results of the experiment

Effect of the TABLE 18 Polypeptides on the mortality of C.elegans

Group of	Dosage of polypeptide (μ M)	Mortality (%)
			Control group	/	1.69±0.52
Polypeptide low dose group	5	1.39±0.77#
			Polypeptide medium dose group	10	1.89±0.67#
High dose group of polypeptides	20	1.42±0.74#

Note: ratio # P >0.05 to blank control.

To investigate the acute toxicity of anti-CTLA-4 polypeptides, the effect of the polypeptides on the mortality of C.elegans was mainly evaluated. Compared with a blank control group, the anti-CTLA-4 polypeptide administration group has no significant difference compared with the control group, and the anti-CTLA-4 polypeptide has no influence on the mortality of the caenorhabditis elegans under the experimental dosage and has better safety.

Example 8

Detection of Chronic toxicity of anti-CTLA-4 Polypeptides to C.elegans

1 materials of the experiment

N2 wild type caenorhabditis elegans, CTLA-4 resisting polypeptide, a culture dish, polypeptone, agar powder and sodium hypochlorite.

2 method of experiment

OP50 E.coli was cultured and transferred every other day to 150mL of LB medium for culture. Placing caenorhabditis elegans on OP50 Escherichia coli liquid, controlling room temperature at 20 deg.C for synchronization of caenorhabditis elegans, and allowing larva to stay in L1 stage by cracking into adult and controlling nutrition. The administration group was set to three concentrations of 5. mu.M, 10. mu.M and 20. mu.M, and 50. mu.LOP 50 E.coli strain and 40L 1 larvae (50. mu.L) were added to each well of a 96-well plate. Blank group 100. mu.L/well M9 buffer was added to the experimental group 100. mu.L/well anti-CTLA-4 polypeptide (5. mu.M, 10. mu.M, 20. mu.M) was added according to a dose gradient. Each set of 4 duplicate wells and ensuring a final volume of 200 μ L per well. And after the dosing treatment is finished, culturing for 48 hours in a constant-temperature incubator, and after 48 hours, selecting 15 nematodes in each hole and recording the number of times of head swinging of each nematode within 1 min. (swinging the head to the other side and back to the original position once). Statistical analysis was performed using the SPSS17.0 mathematical statistics software package, mean + -SD. Group comparisons were by T-test with significant statistical differences at P < 0.05.

3 results of the experiment

Effect of the TABLE 19 Polypeptides on the frequency of oscillation of the head

Group of	Dosage of polypeptide (μ M)	Number of head swings (/ min)
			Control group	/	36±0.52
Polypeptide low dose group	5	32±0.77#
			Polypeptide medium dose group	10	36±0.67#
High dose group of polypeptides	20	37±0.74#

Note: ratio # P >0.05 to blank control.

To investigate the chronic toxicity of anti-CTLA-4 polypeptides, the effect of the polypeptides on the frequency of C.elegans head wobble was mainly evaluated. Compared with a blank control group, the anti-CTLA-4 polypeptide administration group has no significant difference compared with the control group, and the anti-CTLA-4 polypeptide basically has no influence on the head swinging frequency of the caenorhabditis elegans under the experimental dosage and has better safety.

Sequence listing

<110> university of Chinese pharmacy

<120> polypeptide with immune checkpoint CTLA-4 inhibitory activity and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>10

<212>PRT

<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)

<400>1

Lys Met Val Leu Thr Met Met Ser Gly Asp

1 5 10

Claims (4)

1. A polypeptide having immune checkpoint CTLA-4 inhibitory activity, wherein: having KX_aVLTX_bMSGD or a pharmaceutically acceptable salt thereof, wherein X_a、X_bIs an amino acid which may be deleted or substituted.

2. The polypeptide of claim 1, wherein: the polypeptide sequence is a polypeptide which still has the function of targeting CTLA-4 after one or more amino acids are deleted, substituted or added on the basis of the sequence of claim 1, or a pharmaceutically acceptable salt of the polypeptide.

3. The polypeptide of claim 1, wherein the amino acid sequence of said polypeptide is KMVLTMMSGD.

4. Use of the polypeptide according to any one of claims 1-3 in the preparation of a medicament for the prevention and treatment of tumors.

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