CN111533782A

CN111533782A - Active polypeptide of targeting immune checkpoint LAG-3 and application thereof

Info

Publication number: CN111533782A
Application number: CN202010380701.8A
Authority: CN
Inventors: 徐寒梅; 夏春磊; 刘晨
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-08-14

Abstract

The invention discloses a LAG-3 targeted polypeptide with anti-tumor activity and application thereof in tumors, belongs to the field of medicaments for treating tumors, and particularly relates to a LAG-3 targeted polypeptide and application thereof in the aspect of anti-tumor. The polypeptide has an amino acid sequence Val-Glu-Xa-Thr-Xb-Arg-Ser or a pharmaceutically acceptable salt thereof, wherein Xa and Xb are any amino acid substitution or deletion. The polypeptide is known to have definite target through a large number of experiments, in vitro and in vivo experiments show that the sequence polypeptide can be specifically combined with LAG-3 positive cells, and further in vitro co-incubation experiments and mouse tumor-bearing experiments show that the polypeptide has better application prospects in the aspects of tumor treatment and tumor-bearing mouse survival improvement, and provides new possibility for immunotherapy of biological tumors. The LAG-3 targeting peptide designed by the invention is scientific, reasonable, feasible and effective, and can be used for preparing a therapeutic medicament for treating human tumors.

Description

Active polypeptide of targeting immune checkpoint LAG-3 and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an active polypeptide targeting LAG-3 and application thereof in the aspect of tumor resistance.

Background

The prior treatment methods mainly comprise surgical operation, chemotherapy and radiotherapy aiming at the tumor diseases, and the methods have certain effects but have certain defects and shortcomings. With the intensive scientific research, we have now entered a new era of immunotherapy. Immunotherapy is achieved by modulating the immune system, especially T cell function, in a manner that activates or induces tumor patients to mount a specific immune response to tumor antigens, eliminates primary tumor cells, and builds up immune memory, preventing tumor recurrence and metastasis, and in addition, the therapy is not only effective, but also avoids damage to the body. Overall, tumor immunotherapy is significantly different from previous surgery, radiation therapy and chemotherapy for tumors. Although tumor immunotherapy is the current research focus, due to the diversity and complexity of tumor types, tumor immunotherapy has also been a difficult point of research.

The existing tumor immunotherapy mainly has two directions, namely, a target tumor cell and an activated immune cell. In the treatment of target tumor cells, bacterial toxins, oncolytic viruses and monoclonal antibodies are generally used for treatment, but the first two have high toxicity and uncontrollable property, so that the monoclonal antibodies are generally used for targeting tumors at present. Among the treatments for activating immune cells, there are mainly four: (1) cell therapy, mainly by activating T cells using cytokine stimulation to restore the function of killing tumor cells, such as CAR-T therapy; (2) tumor vaccine, which can activate human body immune system to eliminate tumor by injecting tumor antigen into human body; (3) an agonist of the immune system, which enhances the immune system of the body by administering to the body an amount of the cytokine IL-2 or the immune adjuvant BCG. (4) The immune checkpoint inhibitor can generally identify and kill tumor cells by the immune system of the body, but the tumor cells can escape by reducing the immunogenicity of self-antigens or inducing immune suppression of the body, thereby avoiding the monitoring of the immune system of the body. Depending on the effect produced, immune checkpoint molecules can be classified as positive co-stimulatory molecules and negative co-stimulatory molecules. Among the negative co-stimulatory molecules, molecules such as CTLA-4, PD-1, and LAG-3 have been more and more important.

Lymphocyte activation gene-3 (LAG-3, CD223), an important negative co-stimulatory molecule, is a membrane protein of a member of the immunoglobulin superfamily, which is activated CD4⁺And CD8⁺T cells and a proportion of Natural Killer (NK) cells are upregulated. LAG-3 protein molecules consist of 525 amino acids and are bound to major histocompatibility complex class II molecules (MHC-II) primarily via extracellular IgV-like domains. LAG-3 expression can make T cell in depletion state, maintain self and tumor antigen immune tolerance, and anti-LAG-3 monoclonal antibody can block LAG-3 molecular function, thereby reversing inhibited T cell activity and function.

Aiming at LAG-3, the existing research shows that the interaction of LAG-3 and ligand MHC-II thereof is an important negative co-stimulatory molecule in the T cell activation process, and plays an important role in inducing the incapability of T cells and the maintenance of immune tolerance, thereby mediating the immune tolerance and escape of tumors. Therefore, the target substance designed aiming at the target can avoid or reduce the negative co-stimulation effect of the LAG-3 to a certain extent, and has very important significance for the field of tumor immunotherapy.

Disclosure of Invention

1. Problems to be solved

The invention mainly aims to provide a polypeptide with a brand-new sequence, which is characterized in that the polypeptide can better target LAG-3, the sequence of the polypeptide is Val-Glu-Xa-Thr-Xb-Arg-Ser, or pharmaceutically acceptable salt thereof, wherein Xa and Xb are any amino acid substitution or deletion. After the series of polypeptides are further selected to carry out corresponding animal experiments, the polypeptides are proved to be capable of better inhibiting the tumor growth of tumor-bearing mice, and the polypeptides are shown to have certain potential for tumor immunotherapy.

2. Technical scheme

A polypeptide targeting LAG-3, characterized in that: the amino acid sequence of the polypeptide is Val-Glu-Xa-Thr-Xb-Arg-Ser or pharmaceutically acceptable salt thereof, wherein Xa and Xb are any amino acid substitution or deletion.

The polypeptide amino acid sequence has any one of the following amino acid sequences:

Val-Glu-Thr-Thr-Phe-Arg-Ser(VS-7-1)、Val-Glu-Ala-Thr-Phe-Arg-Ser(VS-7-2)、

Val-Glu-Thr-Thr-Ala-Arg-Ser(VS-7-3)、Val-Glu-Thr-Phe-Arg-Ser(VS-7-4)、

Val-Glu-Thr-Thr-Arg-Ser(VS-7-5)。

the polypeptide can be used as a medicament for preventing and/or treating tumors, and the active component of the polypeptide is the polypeptide with the sequence.

The LAG-3-targeting polypeptide is used for preparing a medicament for preventing or treating tumors, such as: esophageal cancer, breast cancer, ovarian cancer, colon cancer, leukemia, gastric cancer, lung cancer, melanoma, renal cancer, and classical hodgkin's lymphoma.

If necessary, one or more pharmaceutically acceptable adjuvants can be added into the above medicine, and the adjuvants include diluent, filler, binder, wetting agent, absorption enhancer, surfactant, lubricant or stabilizer conventional in pharmaceutical field.

The medicine of the present invention may be prepared into injection, dry powder injection and other forms. The medicines of the above dosage forms can be prepared according to the conventional method in the conventional pharmaceutical field.

The current clinical immune checkpoint inhibitor is mainly monoclonal antibody, but antibody drugs have certain defects and shortcomings in the using process, such as high cost, high immunogenicity, complex production process and the like.

The action mechanism is as follows:

aiming at the targeted LAG-3 polypeptide, the targeted LAG-3 polypeptide is proved to be better combined with LAG-3 positive cells in vitro; further in vitro tests prove that the recombinant human interferon can well reactivate the inhibited immune cells and promote the secretory release of IL-2 and IFN-gamma; the mouse tumor-bearing test shows that the compound can better inhibit the growth of tumors. The results show that the polypeptide has better application prospect in the aspect of tumor resistance, and provides new possibility and method for immunotherapy of biological tumors.

3. Advantageous effects

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

(1) the polypeptide of the invention has simple structure, and is easy to synthesize, separate and purify;

(2) the polypeptide of the invention can effectively reactivate the inhibited immune cells and promote the secretion and release of IL-2 and IFN-gamma;

(3) the polypeptide can better eliminate the immunosuppressive effect in vitro and in vivo, and has the specific effects of obviously improving the activity of T cells and preventing the tumor growth of tumor-bearing mice;

(4) the polypeptide has no obvious adverse reaction and toxic and side effect.

Drawings

FIG. 1 is a chart showing the purity of each polypeptide by HPLC. Wherein, the graph A is a VS-7-1 purity result graph, the graph B is a VS-7-2 purity result graph, the graph C is a VS-7-3 purity result graph, the graph D is a VS-7-4 purity result graph, and the graph E is a VS-7-5 purity result graph.

FIG. 2 is a flow cytometer used to detect the binding of T cells to FITC-VS-7-1. Where panel A is a negative control and B, C is the binding rate of T cells to 1. mu.M, 10. mu.M FITC-conjugated VS-7-1 polypeptide.

Results are expressed as mean ± SD. The comparison between groups was performed by T-test, where P <0.05 was significant statistical significance for the difference and P <0.01 was very significant statistical significance for the difference.

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. Any simple modifications or equivalent changes made to the following examples according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Val-Glu-Thr-Thr-Phe-Arg-Ser (VS-7-1), Val-Glu-Ala-Thr-Phe-Arg-Ser (VS-7-2), Val-Glu-Thr-Thr-Ala-Arg-Ser (VS-7-3), Val-Glu-Thr-Phe-Arg-Ser (VS-7-4) and Val-Glu-Thr-Thr-Arg-Ser (VS-7-5) are biochemically synthesized by Shanghai Jier, and the purity is more than 95%.

Example 1

The polypeptides involved in this example were all obtained by solid phase synthesis and then purity checked by high performance liquid HPLC.

The polypeptide sequences are as follows: the purities of Val-Glu-Thr-Thr-Phe-Arg-Ser (VS-7-1), Val-Glu-Ala-Thr-Phe-Arg-Ser (VS-7-2), Val-Glu-Thr-Thr-Ala-Arg-Ser (VS-7-3), Val-Glu-Thr-Phe-Arg-Ser (VS-7-4) and Val-Glu-Thr-Thr-Arg-Ser (VS-7-5) are more than 95%. The results are shown in FIG. 1. In FIG. A, the purity of VS-7-1 was 98.98%, in FIG. B, the purity of VS-7-2 was 98.76%, in FIG. C, the purity of VS-7-3 was 99.76%, in FIG. D, the purity of VS-7-4 was 99.08%, in FIG. E, the purity of VS-7-5 was 97.17%.

Example 2 specific binding assay of targeted LAG-3 polypeptides to targets at the cellular level.

In this example, the VS-7-1 polypeptide was mainly used as a target for the study of polypeptides for preventing and/or inhibiting tumor activity.

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 main target of an active polypeptide for preventing and/or treating tumors, which is LAG-3, wherein a VS-7-1 polypeptide is connected with a FITC fluorescent marker, and the binding capacity of the polypeptide with cell surface LAG-3 at the cell level is reflected by the binding rate of cells and a FITC-VS-7-1 polypeptide solution.

1.1 Experimental materials

CD3⁺T cells, BSA blocking solution, Ficoll kit, CD3 sorting magnetic beads, CD3/CD28 activation magnetic beads.

1.2 Experimental instruments

CO₂Cell culture case, single channel pipettor, inverted microscope, flow cytometer, liquid nitrogen tank, horizontal centrifuge, small centrifuge, superclean bench, full-automatic high pressure steam sterilization pot, oven, electronic balance.

1.3 Experimental methods

According to the kitBook for reading CD3⁺Sorting and activating T cells, and adding 1640 culture solution containing CD3⁺Collecting T cells, washing twice with ice-precooled PBS, adding 1mL of 1% BSA solution, fixing on a rotary mixer, mixing for 30min at 4 ℃, adding a corresponding amount of FITC-coupled VS-7-1 polypeptide solution under the condition of light shielding, wherein the final concentrations are 1 mu M and 10 mu M respectively, fixing on the rotary mixer, mixing for 1h at 4 ℃ in the light shielding condition, after finishing incubation of the drug, 800rpm, centrifuging for 5min, removing the supernatant, washing twice with precooled PBS, and adjusting the cell concentration to 1 × 10 by PBS⁶cells/mL. Duplicate wells of 3 parallel wells were made for each group of cells and 0.5mL of each sample was prepared.

Binding of the polypeptide to LAG-3 positive cells was detected using flow cytometry. And (3) sequentially turning on the stabilized voltage power supply, the transformer, the flow cytometer host, the computer and the 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. Air bubbles are removed between the liquid flow line and the filter. The sample tube was removed and PRIME function was performed twice, 1mL facclean. 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 vacuumed, the sample holder was back up, 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 flow cytometer was turned off after allowing to work for 20min according to Standby. And exiting the program and closing the computer.

The results are shown in FIG. 2, which shows that the VS-7-1 polypeptide binds well to LAG-3 positive cells.

Example 3

Detecting the regulation and control of the series of target LAG-3 polypeptides on cytokines in a tumor cell and T cell co-incubation system. The ELISA kit detects the secretion of IL-2 and IFN-gamma in the co-incubation system, and the related experimental conditions are described as follows.

2.1 materials of the experiment

CD3⁺T cell, regulation and control esophageal cancer cell KYSE-30, breast cancer cell MDA-MB-231, ovarian cancer cell A2780, colon cancer cell HCT-116 and leukemiaThe cell Jurkat, the gastric cancer cell MGC-803, the lung cancer cell A549, the melanoma cell ME, the renal cancer cell A-498, the typical Hodgkin lymphoma cell L428, the tumor cells are all from ATCC, Ficoll kit, CD3 sorting magnetic beads, CD3/CD28 activating magnetic beads, IL-2 cell factors, IL-2ELISA detection kit and IFN-gamma detection kit, U-shaped 96-pore plate, RPMI 1640 culture medium.

2.2 Experimental instruments

An inverted microscope, a liquid nitrogen tank, a horizontal centrifuge, a small-sized centrifuge, a super clean bench, a full-automatic high-pressure steam sterilization pot, an oven, an electronic balance and an automatic cell counter.

2.3 Experimental methods

CD3 according to kit instructions⁺Sorting and activating T cells, and adding CD3 containing 1640 culture solution⁺T cells were collected and washed twice with ice-cold PBS. Collection at 37 ℃ with 5% CO₂The culture chamber of (2) was used to culture various tumor cells in logarithmic growth phase, the cell concentration was adjusted to the desired concentration, and the cells were inoculated in a U-shaped 96-well plate. Mixing T cells and tumor cells at different ratio, and adding 5% CO at 37 deg.C₂The culture box is cultured for 24h, and the optimal co-incubation proportion is screened by measuring the secretion of the cytokines IFN-gamma and IL-2.

Mixing T cells and tumor cells at the optimal CO-incubation ratio selected above, wherein the control group is a group without polypeptide, the positive control Anti-LAG-3 (from BioLegend) is a group with different concentrations of the target polypeptide, and the test group is a group with 5% CO at 37 deg.C₂The culture chamber of (1) was incubated for 24 hours, and then the secretion amounts of the cytokines IFN-. gamma.and IL-2 were measured by ELISA method. The effect of the targeting polypeptide on LAG-3/MHC II signaling pathway of lymphocyte effector cells is reflected by the change of the secretion amount of the above-mentioned cytokine in the co-incubation system. Results were statistically analyzed using SPSS 19.0(SPSS inc. chicago, IL, USA) software.

The results are shown in tables 1 and 2, and it is shown in the tables that each targeted LAG-3 polypeptide can reverse the T cell suppression state to some extent, restore or reactivate T cells, and promote secretion of IL-2 and IFN- γ.

TABLE 1 polypeptide regulation of IL-2 levels in tumor cell and T cell co-incubation systems

TABLE 2 Each polypeptide regulates IFN-gamma level in co-incubation system of tumor cells and T cells

Example 4

The present example mainly performed in vivo animal experiments on the antitumor activity of the targeted LAG-3 polypeptides as analytes, and the related experimental procedures are described below.

3.1 Experimental materials

Balb/c nude mice, targeted LAG-3 series polypeptides, Ficoll kit, CD3 cell sorting magnetic beads, CD3/CD28 activated magnetic beads, esophageal cancer cells KYSE-30, breast cancer cells MDA-MB-231, ovarian cancer cells A2780, colon cancer cells HCT-116, leukemia cells Jurkat, stomach cancer cells MGC-803, lung cancer cells A549, melanoma cells ME, kidney cancer cells A-498, classical Hodgkin lymphoma cells L428, IL-2 cytokines, RPMI 1640 culture medium and calipers.

3.2 Experimental instruments

The system comprises an inverted microscope, a liquid nitrogen tank, a horizontal centrifuge, a small-sized centrifuge, a super clean bench, a full-automatic high-pressure steam sterilization pot, an oven, an electronic balance, an automatic cell counter and a small animal living body imager.

3.3 Experimental methods

Healthy volunteers were tested for tumor suppression rate in their blood using esophageal cancer cell KYSE-30, breast cancer cell MDA-MB-231, ovarian cancer cell A2780, colon cancer cell HCT-116, leukemia cell Jurkat, gastric cancer cell MGC-803, lung cancer cell A549, melanoma cell ME, renal cancer cell A-498 and Balb/c nude mice with classical Hodgkin lymphoma cell L428. subcutaneous injection 2 × 10 was made in the lateral axilla of each mouse⁶Tumor cells and 5 × 10⁵Human P activated by CD3/CD28 activated magnetic beadsBMC cells, total volume 0.1mL, were then randomly divided into four groups. The in vivo test corresponding to each tumor is divided into 4 groups, namely a Control group (Control), a target polypeptide high-dose group (4mg/kg), a target polypeptide medium-dose group (2mg/kg), a target polypeptide low-dose group (1mg/kg) and four groups, wherein each group comprises 8-10 Balb/c nude mice.

When in administration, the polypeptide is dissolved in normal saline to prepare polypeptide drugs with corresponding concentration, and the polypeptide drugs are subpackaged and stored at 20 ℃ below zero for later use.

Subcutaneous administration was performed for 21 days for each group. Mice were free to eat and drink water during the experiment. After the administration was stopped, the mice were observed for survival.

The weight change of the series of targeted peptide group mice in the experimental period is in a normal range, and the primary indication that the targeted peptide has no obvious toxic or side effect and adverse reaction can be realized.

The results are as follows, see table 3: the target LAG-3 series polypeptide can obviously inhibit the tumor growth of various tumor-bearing mice within a certain concentration range, and shows certain dose dependence.

TABLE 3 growth inhibition ratio (%)

Sequence listing

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Claims

1. A polypeptide targeting LAG-3, characterized in that: the amino acid sequence of the polypeptide is Val-Glu-Xa-Thr-Xb-Arg-Ser or pharmaceutically acceptable salt thereof, wherein Xa and Xb are any amino acid substitution or deletion.

2. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide is any one of:

Val-Glu-Thr-Thr-Phe-Arg-Ser、Val-Glu-Ala-Thr-Phe-Arg-Ser、

Val-Glu-Thr-Thr-Ala-Arg-Ser, Val-Glu-Thr-Phe-Arg-Ser, or Val-Glu-Thr-Thr-Arg-Ser.

3. Use of a polypeptide according to claim 1 or 2 for the preparation of a medicament for the prevention or treatment of cancer.

4. Use according to claim 3, characterized in that: the cancer is esophageal cancer, breast cancer, ovarian cancer, colon cancer, leukemia, gastric cancer, lung cancer, melanoma, renal cancer or classical Hodgkin lymphoma.

5. A pharmaceutical composition comprising the polypeptide of claim 1 or 2, and a pharmaceutically acceptable excipient.

6. A pharmaceutical composition according to claim 5, wherein said excipient comprises a diluent, filler, binder, wetting agent, absorption enhancer, surfactant, lubricant or stabilizer.

7. The pharmaceutical composition of claim 6, wherein the composition is formulated as an injection solution or a lyophilized powder for injection.