Multi-specific peptide nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a multispecific peptide nanoparticle as well as a preparation method and application thereof, and belongs to the technical field of medicines. The multi-specific peptide nano-particles are nano-particles formed by mixing and co-assembling an amphiphilic polymer modified by a tumor cell targeting peptide, an amphiphilic polymer modified by a T cell targeting peptide and an unmodified amphiphilic polymer or / and an amphiphilic phospholipid molecule, the tumor cell targeting peptide and the T cell targeting peptide are both modified at the hydrophilic end of the amphiphilic polymer. The multi-specific peptide nanoparticles provided by the invention promote the combination of tumor cells and T cells and enhance the killing effect of the T cells on the tumor cells, have no obvious toxic or side effect, and have good clinical transformation prospects and practical significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a multispecific peptide nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Bispecific antibody (bsAb) is an antibody molecule containing two independent target binding fragments with specific epitopes, which has multi-targeting and multi-functionality compared with single antibody. For example, Blinatumomab is a bispecific antibody used for treating relapsed B-cell acute lymphoblastic leukemia, which specifically bridges T cells and malignant B cells by simultaneously targeting T cell surface receptor CD3 and malignant B cell surface receptor CD19, and activates T cells to kill malignant B cells.

[0003] In the past decade, 308 clinical studies related to bispecific antibodies have been conducted, including 126 bispecific antibody drugs, and the number of clinical trials for treating hematological tumors and solid tumors is comparable. In the bispecific antibody clinical trials for treating hematological tumors, more than half of the tumor cells target CD19, followed by CD20, BCMA and CD123, and most of the immune cells target CD3 of T cells. In the bispecific antibody clinical trials for treating solid tumors, tumor cells target HER2, EGFR and PD-L1, and immune cells target PD-1, CTLA-4 and CD3. In addition to the common T cell bridging bispecific antibodies, the types of bispecific antibody drugs are also continuously updated and optimized, including immune checkpoint blockade-tumor associated antigen inhibition type, immune checkpoint blockade-tumor microenvironment regulation type, double immune checkpoint blockade type, double tumor associated antigen inhibition type and promotion of functional complex protein formation type, etc.

[0004] Although the number of clinical trials of new bispecific antibody drugs continues to grow, bispecific antibodies still face some challenges. First, the use of antibody therapy may cause cytokine storm (CRS) and other safety problems, although some methods have been developed to reduce the risk of CRS, such as selecting a weaker affinity CD3 binding unit, adjusting the distance between the two antibodies or changing the target point, but these methods may reduce the efficacy. Second, due to heterogeneity and randomness, it is easy to mismatch between light and heavy chains, although various modification methods are designed to separate the light and heavy chains of the two antibodies to reduce mismatching, but the modification of antibodies increases the process steps and preparation time. In addition, due to the complex structure of bispecific antibodies, the drugability is low, and protein fragmentation and aggregation may also occur, making it difficult to establish a unified method for studying the activity of bispecific antibody drugs.

[0005] Compared with therapeutic antibodies, polypeptides have the advantages of low immunogenicity, low cost, high purity, good tissue penetration, easy to synthesize and modify, and good safety, and can inhibit the occurrence and development of tumors by activating the body's anti-tumor immune response, inhibiting tumor neovascularization, and inducing tumor cell apoptosis and necrosis. However, polypeptides have the defects of small molecular weight, poor in-vivo stability, and short half-life.

[0006] By chemically modifying polypeptides on the surface of nanoparticles, the hydrodynamic radius of the polypeptides can be increased, thereby reducing the filtration of the glomerulus, prolonging the in-vivo circulation half-life, and increasing the amount of polypeptides to improve the affinity for the target. Simultaneously modifying tumor cell targeting peptides and T cell targeting peptides on the nanoparticles can endow the nanoparticles with the ability to specifically target and bridge tumor cells and T cells, thereby enhancing the killing of tumor cells by T cells. SUMMARY

[0007] The purpose of the present application is to couple several polypeptide molecules that specifically target tumor cells and T cells on nanoparticles to construct multispecific peptide nanoparticles, thereby retaining the functional activity of single-target polypeptides while improving the stability and affinity of the polypeptides for the target, specifically targeting and bridging tumor cells and T cells, mediating the killing of tumor cells by T cells, and enhancing the immunotherapy effect on tumors.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The present application provides a multispecific peptide nanoparticle, which is a nanoparticle formed by co-assembly of a tumor cell targeting peptide modified amphiphilic polymer, a T cell targeting peptide modified amphiphilic polymer, and an unmodified amphiphilic polymer or / and an amphiphilic phospholipid molecule, wherein the tumor cell targeting peptide and the T cell targeting peptide are both modified on the hydrophilic end of the amphiphilic polymer.

[0009] In the present application, amphiphilic molecules self-assemble into nanoparticles with a hydrophobic core and a hydrophilic shell in an aqueous environment through intramolecular and intermolecular hydrophobic forces. In the present application, polypeptide molecules that target tumor cells and polypeptide molecules that target T cells are modified on the hydrophilic end of the amphiphilic polymer, respectively. After self-assembly, several polypeptide molecules are modified on the surface of the nanoparticles, thereby endowing the nanoparticles with the ability to specifically target and bridge tumor cells and T cells, and mediating the killing of tumor cells by T cells. Each nanoparticle is modified with multiple polypeptides that target tumor cells and polypeptides that target T cells, and the multivalent effect can enhance the interaction between the nanoparticles and the cells, thereby enhancing the connection between the tumor cells and the T cells.

[0010] The present application mixes the amphiphilic polymer coupled with the targeting peptide with the amphiphilic polymer or / and the amphiphilic phospholipid molecule without modification to prepare the micellar nanoparticle, and the addition of the amphiphilic polymer or / and the amphiphilic phospholipid molecule without modification can stabilize the structure of the nanoparticle.

[0011] Therefore, the present application constructs a multi-specific peptide nanoparticle platform which can be simply and efficiently prepared, and realizes multivalency and multi-specificity.

[0012] As preferred, the hydrophilic end of the amphiphilic polymer is modified with a maleimide, and the terminal of the targeting peptide is modified and connected to the hydrophilic end of the amphiphilic polymer through a thiol group.

[0013] As preferred, the amphiphilic polymer is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG). DSPE-PEG has a clear and controllable molecular structure, and can be used as a polypeptide display skeleton by functional modification of the molecular structure through chemical reaction.

[0014] More preferably, the molecular weight of the polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol is 2000. The peptide nanoparticle formed by using the DSPE-PEG with the molecular weight has good stability.

[0015] As preferred, the amphiphilic phospholipid molecule is phosphatidylcholine (PC).

[0016] The polypeptide targeting tumor cells can specifically bind to tumor cells, and the polypeptide targeting T cells can specifically bind to T cells. As a specific embodiment of the present application, the present application provides a bispecific peptide nanoparticle against PD-L1 and CD3, wherein the tumor cell targeting peptide in the bispecific peptide nanoparticle is a polypeptide targeting PD-L1, and the T cell targeting peptide is a polypeptide targeting CD3. The polypeptide targeting PD-L1 can specifically bind to tumor cells overexpressing PD-L1, and the polypeptide targeting CD3 can specifically bind to T cells.

[0017] As preferred, the nanoparticle is formed by self-assembly of the amphiphilic polymer modified with the polypeptide targeting PD-L1, the amphiphilic polymer modified with the polypeptide targeting CD3, and the amphiphilic polymer or / and the amphiphilic phospholipid molecule without modification in a molar ratio of 1:1:3-6. The molar ratio of the amphiphilic polymer without modification and the amphiphilic phospholipid molecule is 1:1.

[0018] As preferred, the amino acid sequence of the polypeptide targeting PD-L1 is CNYSKPTDRQYHF (as shown in SEQ ID NO. 1).

[0019] As preferred, the amino acid sequence of the polypeptide targeting CD3 is CAKMGEGGWGANDY (as shown in SEQ ID NO. 2).

[0020] As a specific embodiment of the present application, the present application provides a trispecific peptide nanoparticle against PD-L1, LAG-3 and 4-1BB, wherein the polypeptide targeting tumor cells is a polypeptide targeting PD-L1, and the polypeptide targeting T cells are polypeptides targeting LAG-3 and 4-1BB respectively. The polypeptide targeting PD-L1 can specifically bind to tumor cells overexpressing PD-L1, and both the polypeptide targeting LAG-3 and the polypeptide targeting 4-1BB can specifically bind to T cells.

[0021] As preferred, the nanoparticle is formed by self-assembly of the amphiphilic polymer modified by the polypeptide targeting PD-L1, the amphiphilic polymer modified by the polypeptide targeting LAG-3, the amphiphilic polymer modified by the polypeptide targeting 4-1BB, and the amphiphilic polymer or / and the amphiphilic phospholipid molecule without modification, in a molar ratio of 1:1:1:3-6. The molar ratio of the amphiphilic polymer without modification and the amphiphilic phospholipid molecule is 1:1.

[0022] As preferred, the amino acid sequence of the polypeptide targeting PD-L1 is CVRARTR (as shown in SEQ ID NO. 3).

[0023] As preferred, the amino acid sequence of the polypeptide targeting LAG-3 is CMHRPPST (as shown in SEQ ID NO. 4).

[0024] As preferred, the amino acid sequence of the polypeptide targeting 4-1BB is CEKPGGGF (as shown in SEQ ID NO. 5).

[0025] The present application also provides a method for preparing the multispecific peptide nanoparticle, comprising the following steps: (1) coupling tumor cell targeting peptides and T cell targeting peptides to amphiphilic polymers modified with maleimide at the hydrophilic end by chemical reaction; (2) mixing the amphiphilic polymers modified with tumor cell targeting peptides, the amphiphilic polymers modified with T cell targeting peptides, and the amphiphilic polymers without modification or / and the amphiphilic phospholipid molecules, and then self-assembling into micellar nanoparticles in water by solvent injection method or dialysis method to prepare the multispecific peptide nanoparticle.

[0026] In step (1), the maleimide group at the hydrophilic end of the amphiphilic polymer reacts with the sulfhydryl group at the N-terminus of the polypeptide molecule under the catalysis of an organic base, realizing the combination of the two.

[0027] As preferred, the organic base can be, but not limited to, triethylamine (TEA), N, N-diisopropylethylamine (DIPEA).

[0028] As preferred, the terminal of the polypeptide molecule is cysteine (Cys).

[0029] As preferred, the reaction medium is dimethyl sulfoxide.

[0030] As preferred, in the reaction system, the concentration of the amphiphilic polymer is 4 mmol / L calculated by the modified maleimide, and the molar ratio of the amphiphilic polymer to the polypeptide molecule is 1:1.1.

[0031] As preferred, the reaction is normal temperature reaction for 24 h in nitrogen atmosphere.

[0032] Specifically, the maleimide-modified amphiphilic polymer and the polypeptide molecule are respectively dissolved in anhydrous DMSO, the polypeptide solution is added to the amphiphilic polymer solution, then triethylamine is added, and normal temperature reaction for 24 h is carried out in nitrogen atmosphere. After the reaction is completed, the amphiphilic polymer modified with the polypeptide is cut off by dialysis, and the medium is replaced by water. The corresponding product is obtained by freeze-drying.

[0033] In step (2), the unmodified amphiphilic polymer or / and the amphiphilic phospholipid molecule, the amphiphilic polymer modified with the tumor cell targeting peptide, and the amphiphilic polymer modified with the T cell targeting peptide are respectively dissolved in an organic solvent, mixed, and then self-assembled into micellar nanoparticles in water by solvent injection or dialysis.

[0034] Specifically, the unmodified amphiphilic polymer or / and the amphiphilic phospholipid molecule, the amphiphilic polymer modified with the PD-L1 targeting peptide, and the amphiphilic polymer modified with the CD3 targeting peptide are dissolved in anhydrous DMSO, mixed in a molar ratio of 3:1:1, and then the solution is injected into water and shaken to prepare the said bispecific peptide nanoparticles.

[0035] Specifically, the unmodified amphiphilic polymer or / and the amphiphilic phospholipid molecule, the amphiphilic polymer modified with the PD-L1 targeting peptide, the amphiphilic polymer modified with the LAG-3 targeting peptide, and the amphiphilic polymer modified with the 4-1BB targeting peptide are dissolved in anhydrous DMSO, mixed in a molar ratio of 3:1:1:1, and then the solution is injected into water and shaken to prepare the said trispecific peptide nanoparticles.

[0036] As preferred, the volume ratio of anhydrous DMSO to water is 1:99.

[0037] As preferred, the shaking speed is 2500 rpm, and the time is 1 h.

[0038] The method further comprises removing DMSO by dialysis and concentrating to obtain the multispecific peptide nanoparticle.

[0039] The application further provides use of the multispecific peptide nanoparticle in preparation of a tumor immunotherapy drug.

[0040] When the connected peptide segment in the peptide nanoparticle is directed against PD-L1, the tumor is a PD-L1 overexpressing tumor. Specifically, the tumor includes but is not limited to colon cancer, breast cancer.

[0041] The application has the following beneficial effects: (1) The multispecific peptide nanoparticle constructed in the application can be simply and efficiently obtained through several steps, has a clear structure and is clearly characterized. The polypeptide can be prepared in a large scale and high purity through solid-phase synthesis, connected to the hydrophilic end of a commonly used amphiphilic polymer through efficient reaction of maleimide and thiol, and further constructed into a peptide nanoparticle through self-assembly to realize multivalency and multispecificity.

[0042] (2) The multispecific peptide nanoparticle provided in the application is proved by in-vivo and in-vitro experiments to be capable of bridging tumor cells and T cells and enhancing killing of tumor cells by T cells. In in-vivo experiments, the multispecific peptide nanoparticle provided in the application has more remarkable advantages than combined treatment of single polypeptide nanoparticles, has no obvious toxic side effects, and has good clinical transformation prospects. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The figure is a schematic diagram for preparation of the multispecific peptide nanoparticle in the application.

[0044] Figure 2 The figure is a reaction formula for preparation of DSPE-PEG-PDL1pep and DSPE-PEG-CD3pep in Example 1.

[0045] Figure 3 The figure is a nuclear magnetic resonance hydrogen spectrum of DSPE-PEG-MAL.

[0046] Figure 4 The figure is a nuclear magnetic resonance hydrogen spectrum of a PD-L1 targeting peptide (A) and DSPE-PEG-PDL1pep (B).

[0047] Figure 5 The figure is a nuclear magnetic resonance hydrogen spectrum of a CD3 targeting peptide (A) and DSPE-PEG-CD3pep (B).

[0048] Figure 6 The figure is a reaction formula for preparation of DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep in Example 2.

[0049] Figure 7 NMR spectra of PD-L1 targeting peptide (A) and DSPE-PEG-PDL1 pep (B).

[0050] Figure 8 NMR spectra of LAG-3 targeting peptide (A) and DSPE-PEG-LAG-3 pep (B).

[0051] Figure 9 NMR spectra of 4-1BB targeting peptide (A) and DSPE-PEG-4-1BB pep (B).

[0052] Figure 10 Particle size distribution diagram of bi-specific peptide nanoparticles Bi-NP (A) and tri-specific peptide nanoparticles Tri-NP (B) in water measured by dynamic light scattering.

[0053] Figure 11 Laser confocal observation of the interaction between tumor cells and T cells mediated by bi-specific peptide nanoparticles Bi-NP (A) and tri-specific peptide nanoparticles Tri-NP (B).

[0054] Figure 12 Killing of tumor cells by T cells mediated by bi-specific peptide nanoparticles Bi-NP (A) and tri-specific peptide nanoparticles Tri-NP (B) measured by LDH method.

[0055] Figure 13 In the tumor inhibition experiment of CT26 colon cancer cell-bearing Balb / c mice by bi-specific peptide nanoparticles Bi-NP, the body weight change curve (A), tumor growth curve (B), tumor weight statistical diagram (C) and tumor photos (D) are shown.

[0056] Figure 14 In the tumor inhibition experiment of CT26 colon cancer cell-bearing Balb / c mice by tri-specific peptide nanoparticles Tri-NP, the body weight change curve (A), tumor growth curve (B), tumor weight statistical diagram (C) and tumor photos (D) are shown. DETAILED DESCRIPTION

[0057] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application, and are not used to limit the application scope. Any modification or replacement of the method, step or condition of the application without departing from the spirit and essence of the application shall fall within the scope of the application.

[0058] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0059] DSPE-PEG2000-MAL, purchased from Shanghai Bensuo Biotechnology Co., Ltd., the structural formula is as follows: .

[0060] PC, purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., the structural formula is as follows: .

[0061] The amino acid sequence of the PD-L1 targeting peptide 1 is CNYSKPTDRQYHF; the amino acid sequence of the CD3 targeting peptide is CAKMGEGGWGANDY; the amino acid sequence of the PD-L1 targeting peptide 2 is CVRARTR; the amino acid sequence of the LAG-3 targeting peptide is CMHRPPST; and the amino acid sequence of the 4-1BB targeting peptide is CEKPGGGF, which is synthesized by a biological company. The amino acid sequence from left to right is from N-terminal to C-terminal.

[0062] Example 1 1. Preparation of amphiphilic polymers containing PD-L1 targeting peptide or CD3 targeting peptide (DSPE-PEG-PDL1pep or DSPE-PEG-CD3pep) DSPE-PEG2000-MAL (59.1 mg, 0.02 mmol) was dissolved in 3 mL of anhydrous DMSO, then 2 mL of a PD-L1 targeting peptide 1 (36.5 mg, 0.022 mmol) or CD3 targeting peptide (32.1 mg, 0.022 mmol) anhydrous DMSO solution was added, followed by the addition of anhydrous triethylamine (2.8 μL, 0.02 mmol), and the reaction was carried out at room temperature for 24 h under a nitrogen atmosphere. After the reaction was completed, water dialysis was carried out for 24 h, the dialysis bag had a molecular weight cut-off of 1 KDa, and white powder was obtained by freeze-drying.

[0063] Under the catalysis of triethylamine, the maleimide group on the hydrophilic end of DSPE-PEG2000-MAL and the thiol group on the N-terminal of the polypeptide undergo addition reaction, and the reaction process is as shown in Figure 2 .

[0064] The polymers were characterized by nuclear magnetic resonance, as shown in Figures 3-5 , the hydrogen spectrum of the obtained product has peaks corresponding to the H peaks of DSPE-PEG2000 and polypeptide, and the chemical shift and integral are accurate, so it can be known that DSPE-PEG-PDL1pep and DSPE-PEG-CD3pep are successfully prepared.

[0065] 2. Preparation of bispecific peptide nanoparticles (1) DSPE-PEG2000, DSPE-PEG-PDL1pep and DSPE-PEG-CD3pep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the DMSO solutions of the three were mixed according to a molar ratio of DSPE-PEG2000, DSPE-PEG-PDL1pep and DSPE-PEG-CD3pep of 3:1:1, then the mixed DMSO solution was quickly injected into water (the volume ratio of DMSO to water was 1:99), and was vigorously shaken at a speed of 2500 rpm for 1 h, and finally DMSO was removed by ultrafiltration, and concentrated to obtain the bispecific peptide nanoparticle Bi-NP.

[0066] 3. Preparation of bispecific peptide nanoparticles (2) PC, DSPE-PEG-PDL1pep and DSPE-PEG-CD3pep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the DMSO solutions of the three were mixed according to a molar ratio of PC, DSPE-PEG-PDL1pep and DSPE-PEG-CD3pep of 3:1:1, then the mixed DMSO solution was quickly injected into water (the volume ratio of DMSO to water was 1:99), and was vigorously shaken at a speed of 2500 rpm for 1 h, and finally DMSO was removed by ultrafiltration, and concentrated to obtain the bispecific peptide nanoparticle Bi-NP-2.

[0067] 4. Preparation of bispecific peptide nanoparticles (3) DSPE-PEG2000, PC, DSPE-PEG-PDL1pep and DSPE-PEG-CD3pep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the DMSO solutions of the four were mixed according to a molar ratio of DSPE-PEG2000, PC, DSPE-PEG-PDL1pep and DSPE-PEG-CD3pep of 1.5:1.5:1:1, then the mixed DMSO solution was quickly injected into water (the volume ratio of DMSO to water was 1:99), and was vigorously shaken at a speed of 2500 rpm for 1 h, and finally DMSO was removed by ultrafiltration, and concentrated to obtain the bispecific peptide nanoparticle Bi-NP-3.

[0068] 5. Preparation of nanoparticle PDL1-NP DSPE-PEG2000 and DSPE-PEG-PDL1pep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the DMSO solutions of the two were mixed according to a molar ratio of DSPE-PEG2000 to DSPE-PEG-PDL1pep of 4:1, then the mixed DMSO solution was quickly injected into water (volume ratio of DMSO to water was 1:99), and was shaken vigorously at a speed of 2500 rpm for 1 h, and finally DMSO was removed by ultrafiltration, and the nanoparticles PDL1-NP were obtained by concentration.

[0069] 6. Preparation of Nanoparticles CD3-NP DSPE-PEG2000 and DSPE-PEG-CD3pep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the DMSO solutions of the two were mixed according to a molar ratio of DSPE-PEG2000 to DSPE-PEG-CD3pep of 4:1, then the mixed DMSO solution was quickly injected into water (volume ratio of DMSO to water was 1:99), and was shaken vigorously at a speed of 2500 rpm for 1 h, and finally DMSO was removed by ultrafiltration, and the nanoparticles CD3-NP were obtained by concentration.

[0070] Example 2 1. Preparation of amphiphilic polymers containing PD-L1 targeting peptide 2, LAG-3 targeting peptide or 4-1BB targeting peptide (DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep or DSPE-PEG-4-1BBpep) DSPE-PEG2000-MAL (59.1 mg, 0.02 mmol) was dissolved in 3 mL of anhydrous DMSO, then 2 mL of a PD-L1 targeting peptide 2 (18.9 mg, 0.022 mmol), LAG-3 targeting peptide (20.4 mg, 0.022 mmol) or 4-1BB targeting peptide (17.5 mg, 0.022 mmol) anhydrous DMSO solution was added, followed by the addition of anhydrous triethylamine (2.8 μL, 0.02 mmol), and the reaction was carried out at room temperature for 24 h under a nitrogen atmosphere. After the reaction was completed, water was dialyzed for 24 h, the dialysis bag had a molecular weight cut-off of 1 KDa, and white powder was obtained by freeze-drying.

[0071] Under the catalysis of triethylamine, addition reaction occurred between the maleimide groups on the hydrophilic end of DSPE-PEG2000-MAL and the thiol groups on the N-terminus of the polypeptide, and the reaction process was as shown in Figure 6 .

[0072] The polymers were characterized by nuclear magnetic resonance, as shown in Figure 3 , 7The hydrogen spectrum of the obtained product is shown in Figure 9. The peaks of the hydrogen spectrum of the obtained product correspond to the H peaks of DSPE-PEG2000 and the polypeptide, and the chemical shifts and integrals are accurate, so it can be known that DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep are successfully prepared.

[0073] 2. Preparation of multispecific peptide nanoparticles (1) DSPE-PEG2000, DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep are respectively dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the molar ratio of DSPE-PEG2000, DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep is 3:1:1:1. The DMSO solutions of the four are mixed, and then the uniformly mixed DMSO solution is quickly injected into water (the volume ratio of DMSO to water is 1:99), and is vigorously shaken at a speed of 2500 rpm for 1 h. Finally, the DMSO is removed by ultrafiltration, and the tri-specific peptide nanoparticles Tri-NP are obtained by concentration.

[0074] 3. Preparation of multispecific peptide nanoparticles (2) PC, DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep are respectively dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the molar ratio of PC, DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep is 3:1:1:1. The DMSO solutions of the four are mixed, and then the uniformly mixed DMSO solution is quickly injected into water (the volume ratio of DMSO to water is 1:99), and is vigorously shaken at a speed of 2500 rpm for 1 h. Finally, the DMSO is removed by ultrafiltration, and the tri-specific peptide nanoparticles Tri-NP-2 are obtained by concentration.

[0075] 4. Preparation of multispecific peptide nanoparticles (3) DSPE-PEG2000, PC, DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the DMSO solutions of the five were mixed according to the molar ratio of DSPE-PEG2000, PC, DSPE-PEG-PDL1pep, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep as 1.5:1.5:1:1:1, then the mixed DMSO solution was quickly injected into water (the volume ratio of DMSO and water was 1:99), and was vigorously shaken at a speed of 2500 rpm for 1 h, and finally the DMSO was removed by ultrafiltration, and was concentrated to obtain the tri-specific peptide nanoparticle Tri-NP-3.

[0076] 5. Preparation of nanoparticle P / L-NP DSPE-PEG2000, DSPE-PEG-PDL1pep and DSPE-PEG-LAG-3pep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the DMSO solutions of the three were mixed according to the molar ratio of DSPE-PEG2000, DSPE-PEG-PDL1pep and DSPE-PEG-LAG-3pep as 4:1:1, then the mixed DMSO solution was quickly injected into water (the volume ratio of DMSO and water was 1:99), and was vigorously shaken at a speed of 2500 rpm for 1 h, and finally the DMSO was removed by ultrafiltration, and was concentrated to obtain the nanoparticle P / L-NP.

[0077] 6. Preparation of nanoparticle P / 4-NP DSPE-PEG2000, DSPE-PEG-PDL1pep and DSPE-PEG-4-1BBpep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, and the DMSO solutions of the three were mixed according to the molar ratio of DSPE-PEG2000, DSPE-PEG-PDL1pep and DSPE-PEG-4-1BBpep as 4:1:1, then the mixed DMSO solution was quickly injected into water (the volume ratio of DMSO and water was 1:99), and was vigorously shaken at a speed of 2500 rpm for 1 h, and finally the DMSO was removed by ultrafiltration, and was concentrated to obtain the nanoparticle P / 4-NP.

[0078] 7. Preparation of nanoparticle L / 4-NP DSPE-PEG2000, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep were dissolved in anhydrous DMSO at a concentration of 100 mg / mL, respectively, and mixed according to a molar ratio of 4:1:1 of DSPE-PEG2000, DSPE-PEG-LAG-3pep and DSPE-PEG-4-1BBpep. The mixed DMSO solution was then rapidly injected into water (volume ratio of DMSO to water was 1:99), and the mixture was shaken vigorously at 2500 rpm for 1 h. Finally, DMSO was removed by ultrafiltration, and the nanoparticles L / 4-NP were obtained by concentration.

[0079] Test Example 1 1. Particle size test of multispecific peptide nanoparticles The particle size of the bispecific peptide nanoparticles Bi-NP prepared in Example 1 was determined by dynamic light scattering, as shown in FIG. Figure 10 (A). The particle size of the nanoparticles Bi-NP prepared by the solvent injection method was about 128.1 nm, and the polydispersity index (PDI) was 0.149.

[0080] The particle size of the trispecific peptide nanoparticles Tri-NP prepared in Example 2 was determined by dynamic light scattering, as shown in FIG. Figure 10 (B). The particle size of the nanoparticles prepared by the solvent injection method was 131.7 nm, and the polydispersity index (PDI) was 0.116.

[0081] In in vivo applications, a suitable particle size can promote the long circulation time of nanoparticles in the blood and facilitate the accumulation of nanoparticles in tumors. The particle size of the nanoparticles prepared in Example 1 and Example 2 is about 130 nm, which can prevent premature kidney clearance and prevent clearance of large particles by the cell clearance system in the liver, and is suitable for in vivo applications.

[0082] 2. Experiment of multispecific peptide nanoparticles mediating the binding of tumor cells and T cells After the CT26 cells were labeled with CFSE and inoculated in a confocal culture dish and incubated overnight, the T cells were labeled with CMTPX and co-cultured with the CT26 cells, and the ratio of CT26 cells to T cells was 1:10. After the cells were exposed to various drug treatment groups (all at a concentration of 100 μM) for 6 h, the suspended unbound T cells were washed away, and the binding of CT26 cells and T cells was observed by laser confocal microscopy.

[0083] Specifically, the experimental group Bi-NP, the control group PBS, PDL1-NP, CD3-NP, Mix-NP (prepared by simply mixing PDL1-NP and CD3-NP at a ratio of 1:1). The results are shown inFigure 11 (A) shows that compared with the control group (PBS, PDL1-NP, CD3-NP, Mix-NP), there are more T cells (red) and CT26 cells (green) in the experimental group (Bi-NP) co-localization phenomenon, which shows that the bi-specific peptide nanoparticles can promote the combination of tumor cells and T cells.

[0084] Specifically, the experimental group Tri-NP, the control group PBS, P / L-NP, P / 4-NP, L / 4-NP, Mix-NP (prepared by simply mixing P / L-NP, P / 4-NP, L / 4-NP in a ratio of 1:1:1). The results are as follows Figure 11 (B) shows that compared with the control group (PBS, P / L-NP, P / 4-NP, L / 4-NP, Mix-NP), there are more T cells (red) and CT26 cells (green) in the experimental group (Tri-NP) co-localization phenomenon, which shows that the bi-specific peptide nanoparticles can promote the combination of tumor cells and T cells.

[0085] 3. Multispecific peptide nanoparticle-mediated T cell killing of tumor cells CT26 cells were inoculated in 96-well plates and incubated overnight, T cells were added and co-cultured with CT26 cells, the ratio of CT26 cells to T cells was 1:10. After 48 h of incubation of the cells exposed to various drug treatment groups (concentration was 100 μM), the T cells were washed away, and the survival rate of CT26 cells was determined by LDH detection kit.

[0086] Specifically, the experimental group Bi-NP, the control group PBS, PDL1-NP, CD3-NP, Mix-NP (prepared by simply mixing PDL1-NP and CD3-NP in a ratio of 1:1). The results are as follows Figure 12 (A) shows that compared with the control group (PBS, PDL1-NP, CD3-NP, Mix-NP), the survival rate of CT26 cells in the experimental group (Bi-NP) is the lowest, which shows that the bi-specific peptide nanoparticles can enhance the killing of T cells on tumor cells.

[0087] Specifically, the experimental group Tri-NP, the control group PBS, P / L-NP, P / 4-NP, L / 4-NP, Mix-NP (prepared by simply mixing P / L-NP, P / 4-NP, L / 4-NP in a ratio of 1:1:1). The results are as follows Figure 12 (B) shows that compared with the control group (PBS, P / L-NP, P / 4-NP, L / 4-NP, Mix-NP), the survival rate of CT26 cells in the experimental group (Tri-NP) is the lowest, which shows that the bi-specific peptide nanoparticles can enhance the killing of T cells on tumor cells.

[0088] 4. In vivo anti-tumor therapy experiments of bispecific peptide nanoparticles Bi-NP Twenty-five Balb / c mice were subcutaneously injected with 500,000 CT26 cells. 3 Around 24 hours after treatment, mice were randomly assigned to five treatment groups (n=5): PBS, PDL1-NP, CD3-NP, Mix-NP, and Bi-NP. The dose was 1000 μM / 200 μL, and the drug was injected through the tail vein once every two days for a total of six doses. During the entire treatment process, the body weight of the mice was recorded every two days, and the tumor growth of each group of mice was monitored with a vernier caliper. The tumor volume (mm) was calculated using the formula 3 ): Tumor volume = (longest diameter) × (shortest diameter) 2 On day 12, the mice were sacrificed and the tumors were removed. The tumor weights of each group were measured and photographed.

[0089] like Figure 13 As shown, tumors in the PBS group grew rapidly. The other control groups (PDL1-NP, CD3-NP, and Mix-NP) showed some inhibitory effects on tumor growth, but there were no significant differences among the control groups. The bispecific peptide nanoparticle (Bi-NP) group exhibited the most pronounced tumor inhibition, with an inhibition rate of 59.9%. This inhibition was statistically significantly different from the PDL1-NP, CD3-NP, and Mix-NP groups, demonstrating a synergistic effect. Tumor weight data and images of tumors obtained after tumor dissection also confirmed the significant anti-tumor effect of the bispecific peptide nanoparticle (Bi-NP). Furthermore, the mice showed no significant changes in body weight throughout the treatment process, confirming the drug's good biosafety.

[0090] 5. In vivo anti-tumor therapy experiments of trispecific peptide nanoparticles Tri-NP Thirty Balb / c mice were subcutaneously injected with 500,000 CT26 cells. 3 Around 24 hours after treatment, mice were randomly assigned to six treatment groups (n=5): PBS, P / L-NP, P / 4-NP, L / 4-NP, Mix-NP, and Tri-NP. The dose was 1000 μM / 200 μL, and the drug was injected through the tail vein once every two days for a total of eight doses. During the entire treatment process, the body weight of the mice was recorded every two days, and the tumor growth of each group of mice was monitored with a vernier caliper. The tumor volume (mm) was calculated using the formula 3 ): Tumor volume = (longest diameter) × (shortest diameter) 2 × 0.5. On day 16, the mice were sacrificed and the tumors were removed. The tumor weights of each group were measured and photographed.

[0091] like Figure 14As shown, the tumor of PBS group grew rapidly, the rest of the control groups (P / L-NP, P / 4-NP, L / 4-NP and Mix-NP) had certain inhibitory effect on tumor growth, the Tri-NP group had the most obvious tumor inhibition effect, the inhibition rate was 88.0%, and there was a significant statistical difference compared with P / L-NP, P / 4-NP, L / 4-NP and Mix-NP groups, and a synergistic effect was produced, and the tumor weight data obtained after the tumor was dissected and the tumor real object graph also confirmed the significant anti-tumor effect of Tri-NP. In addition, during the whole treatment process, the body weight of mice did not change obviously, which confirmed that the drug had good biological safety.

[0092] The above examples are used to explain and illustrate the present application, but not to limit the present application, any modification and change made to the present application within the spirit and protection scope of the claims of the present application, all fall within the protection scope of the present application.

Claims

1. A multispecific peptide nanoparticle, characterized in that The multi-specific peptide nanoparticles are nanoparticles formed by co-assembling an amphiphilic polymer modified with a tumor cell targeting peptide, an amphiphilic polymer modified with a T cell targeting peptide, and an unmodified amphiphilic polymer or / and an amphiphilic phospholipid molecule. The tumor cell targeting peptide and the T cell targeting peptide are both modified at the hydrophilic end of the amphiphilic polymer.

2. The multispecific peptide nanoparticle according to claim 1, wherein The amphiphilic polymer is distearoylphosphatidylethanolamine-polyethylene glycol, the hydrophilic end of which is modified with maleimide, and the end of the targeting peptide is modified with a sulfhydryl group and connected to the hydrophilic end of the amphiphilic polymer; the amphiphilic phospholipid molecule is phosphatidylcholine.

3. The multispecific peptide nanoparticle according to claim 1 or 2, wherein: The tumor cell targeting peptide is a polypeptide targeting PD-L1, and the T cell targeting peptide is a polypeptide targeting CD3; the molar ratio of the amphiphilic polymer modified with the polypeptide targeting PD-L1, the amphiphilic polymer modified with the polypeptide targeting CD3, and the unmodified amphiphilic polymer or / and amphiphilic phospholipid molecules is 1:1:3-6, wherein the molar ratio of the unmodified amphiphilic polymer to the amphiphilic phospholipid molecules is 1:

1.

4. The multispecific peptide nanoparticle according to claim 3, wherein The amino acid sequence of the polypeptide targeting PD-L1 is CNYSKPTDRQYHF; the amino acid sequence of the polypeptide targeting CD3 is CAKMGEGGWGANDY.

5. The multispecific peptide nanoparticle according to claim 1 or 2, wherein: The polypeptide targeting tumor cells is a polypeptide targeting PD-L1, and the polypeptide targeting T cells is a polypeptide targeting LAG-3 and 4-1BB, respectively; the molar ratio of the amphiphilic polymer modified with the polypeptide targeting PD-L1, the amphiphilic polymer modified with the polypeptide targeting LAG-3, the amphiphilic polymer modified with the polypeptide targeting 4-1BB to the unmodified amphiphilic polymer or / and the amphiphilic phospholipid molecules is 1:1:1:3-6, wherein the molar ratio of the unmodified amphiphilic polymer to the amphiphilic phospholipid molecules is 1:

1.

6. The multispecific peptide nanoparticle according to claim 5, wherein The amino acid sequence of the polypeptide targeting PD-L1 is CVRARTR; the amino acid sequence of the polypeptide targeting LAG-3 is CMHRPPST; and the amino acid sequence of the polypeptide targeting 4-1BB is CEKPGGGF.

7. The method for preparing multispecific peptide nanoparticles according to any one of claims 1 to 6, wherein: include: (1) Using chemical reactions, the tumor cell targeting peptide and the T cell targeting peptide are respectively coupled to an amphiphilic polymer modified with maleimide at the hydrophilic end; (2) mixing an amphiphilic polymer modified with a tumor cell targeting peptide, an amphiphilic polymer modified with a T cell targeting peptide, and an unmodified amphiphilic polymer or / and an amphiphilic phospholipid molecule, and then self-assembling in water to form micellar nanoparticles using a solvent injection method or a dialysis method to prepare the multi-specific peptide nanoparticles.

8. The preparation method according to claim 7, wherein In step (1), the reaction medium is dimethyl sulfoxide, and in the reaction system, the concentration of the amphiphilic polymer is 4 mmol / L in terms of modified maleimide, and the molar ratio of the amphiphilic polymer to the polypeptide molecule is 1:1.

1.

9. Use of the multispecific peptide nanoparticles according to any one of claims 1 to 6 in the preparation of tumor immunotherapy drugs.

10. The use according to claim 9, characterized in that The tumor is colon cancer or breast cancer.

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