Liposome T-cell agonist based on nano antibody as well as preparation method and application of liposome T-cell agonist

Abstract

The invention relates to a lipidosome T-cell agonist based on a nano antibody as well as a preparation method and application of the lipidosome T-cell agonist. The preparation method comprises the following steps: dissolving distearoyl phosphatidylcholine, cholesterol succinic acid monoester and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 into an organic solvent; pushing the pretreated product by using a polycarbonate film through a pushing device to obtain the nano-liposome; carrying out addition reaction on the nano antibody and distearoyl phosphatidyl ethanolamine-polyethylene glycol-succinimide ester; and adding the nano-liposome and incubating to obtain the agonist. The invention also relates to the agonist prepared by the method and application of the agonist in preparation of antitumor drugs. The agonist has good stability, targeted binding specificity and antitumor activity, has no toxic effect on human normal cells, provides a new technical means and a new platform for application in antitumor treatment, and has positive clinical application significance.

Description

Technical Field

[0001] The present invention belongs to the field of biocomposite materials, and in particular relates to a liposome T cell agonist based on nano-antibodies, and a preparation method and application thereof. Background Art

[0003] T cell-based immunotherapy mobilizes anti-tumor immune responses and activates the most important and largest "tumor cell killers" in the body's immune system - cytotoxic T lymphocytes to produce highly effective anti-tumor effects. It is a frontier hotspot and important topic in tumor immunotherapy research.

[0004] However, the number of cytotoxic T lymphocytes that can accurately target and infiltrate into the tumor is limited. How to improve the targeting of cytotoxic T lymphocytes, recruit sufficient and highly active cytotoxic T lymphocytes to the tumor site, and enhance their killing effect inside the tumor is the key to improving the anti-tumor effect and is also the core problem faced by tumor immunotherapy research.

[0005] CD3-based T cell agonists bind to the CD3 antigen molecules on the T cell receptor (TCR) complex and the tumor-specific antigens, thereby recruiting T cells to the tumor foci and activating T cells to kill tumors.

[0006] Currently, a variety of CD3-based T cell agonists have been developed for use in clinical research and have shown promising anti-tumor efficacy.

[0007] Among them, bispecific T cell engager (BiTE) is the most common type of therapeutic recombinant fusion protein T cell agonist. However, BiTE molecules have defects such as high cost, short half-life in vivo and poor pharmacokinetic characteristics, which limit their widespread promotion and application.

[0008] With the development of antibody engineering, antibody miniaturization has become a new trend in the research of new antibodies. Among them, Nanobody (Nb) is a single-domain antibody (SdAb) existing in camels, alpacas and sharks, containing the variable region of the antibody heavy chain, with a size of about 5nm×4nm. It is the smallest polypeptide unit with complete antigen binding ability discovered so far and is relatively stable.

[0009] Nanobodies have many advantages in tumor diagnosis and treatment due to their small molecular weight and volume. However, on the other hand, these molecular characteristics will lead to an extremely short plasma half-life of nanobodies, which are easily metabolized and cleared quickly during the physiological process of circulation in the body, which has become a disadvantage that restricts the clinical treatment application of nanobodies. How to select suitable carriers as a tool for transporting nanobodies in the body to help seek benefits and avoid harm and give full play to the advantages of nanobodies is a major challenge faced in deepening the application of nanobodies. Summary of the invention

[0010] In a first aspect, the present invention provides a method for preparing a liposome-based T cell agonist based on a nanobody, wherein the method comprises the following steps:

[0011] (1) dissolving distearoylphosphatidylcholine, cholesterol succinate monoester, and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent to prepare a pretreated product;

[0012] (2) using a microporous filter membrane to push the pretreated product through an extruder to obtain nanoliposomes (Liposomes, Lipo);

[0013] (3) allowing the nanobody and distearoyl phosphatidylethanolamine-polyethylene glycol-succinimide ester to undergo an addition reaction to prepare nanobody micelles;

[0014] (4) adding Lipo to the nanobody micelles and incubating them to prepare a nanobody-based liposome T cell agonist.

[0015] In a second aspect, the present invention provides a liposome T cell agonist based on nanoantibodies, wherein the agonist is prepared by the method described in the first aspect of the present invention.

[0016] In a third aspect, the present invention provides the use of the nano-antibody-based liposome T cell agonist described in the second aspect of the present invention in the preparation of an anti-tumor drug, wherein the drug is used in the following aspects: (1) specifically binding to surface-specific markers of targeted T cells, tumor cells and / or tumor microenvironment cells; and (2) activating T cells to exert anti-tumor effects.

[0017] Compared with the prior art, the present invention has the following technical advantages:

[0018] The nano-antibody-based liposome T cell agonist of the present invention (hereinafter sometimes referred to as CD105 / CD3Nb-Lipo when it contains both CD105Nb and CD3Nb) has a small particle size (eg, about 100 nm), is evenly distributed in a 1×PBS solution, has good dispersibility, and is highly stable.

[0019] The nano-antibody-based liposome T cell agonist of the present invention, such as CD105 / CD3Nb-Lipo, has high binding specificity and binding efficiency with T cells and target cells that highly express CD105 (such as Bel7404, etc.).

[0020] The nano-antibody-based liposome T cell agonist of the present invention, such as CD105 / CD3Nb-Lipo, can significantly stimulate T cell proliferation in vitro, upregulate the expression of T cell surface activation molecule markers CD25 and CD69, and promote the secretion of IFN-γ, TNF-α, and IL-2 by T cells.

[0021] The CD105 / CD3Nb-Lipo of the present invention can mediate T cells to specifically kill tumor cells in vivo and in vitro, and prolong the survival period of an organism (such as a mouse).

[0022] In summary, the nanoantibody-based liposome T cell agonist of the present invention provides a new strategy for the application of cellular immunotherapy in anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Nanobody CD3Nb ( Figure 1 A) and CD105Nb( Figure 1 B) purification and identification. CL stands for cell lysate (CL); FT stands for flow through (FT); W1 to W5 stand for wash 1-5, respectively; Marker stands for gradient molecular weight protein; E1 to E5 stand for elution 1-5, respectively.

[0024] Figure 2 The particle size distribution, potential and PDI value of nanoliposomes are shown. Among them, Lipo means nanoliposome (Liposome); CD105Nb-Lipo means CD105Nb is modified on the surface of nanoliposomes; CD3Nb-Lipo means CD3Nb is modified on the surface of nanoliposomes; CD105 / CD3Nb-Lipo means CD105Nb and CD3Nb are modified on the surface of nanoliposomes at the same time.

[0025] Figure 3 The stability of CD105 / CD3Nb-Lipo in 1×PBS at 4°C and 25°C is shown. “Particle size” means particle size; polymer dispersity index means polymer dispersion index (PDI); electric potential means electric potential; and “Time (Day)” means time (day).

[0026] Figure 4 The toxicity test of CD105 / CD3Nb-Lipo on LO2 cells is shown.

[0027] Figure 5 Flow cytometry analysis of the binding ability of CD105 / CD3Nb-Lipo to T cells and Bel7404 cells is shown.

[0028] Figure 6 It shows that CD105 / CD3Nb-Lipo enhances the proliferation ability of T cells.

[0029] Figure 7 The results show that CD105 / CD3Nb-Lipo induces upregulation of CD25, an activation molecule on the surface of T cells. The ordinate represents the percentage of CD25 positive cells.

[0030] Figure 8 CD105 / CD3Nb-Lipo induces upregulation of CD69, a T cell surface activation molecule. The vertical axis represents the percentage of CD69 positive cells.

[0031] Fig. 9 It showed that CD105 / CD3Nb-Lipo induced upregulation of IFN-γ secretion in T cells.

[0032] Fig.10 It showed that CD105 / CD3Nb-Lipo induced upregulation of IL-2 secretion in T cells.

[0033] Fig.11 It showed that CD105 / CD3Nb-Lipo induced upregulation of TNF-α secretion in T cells.

[0034] Fig.12 It shows that CD105 / CD3Nb-Lipo effectively kills Bel7404 cells, wherein the vertical axis represents specific cytotoxicity.

[0035] Fig.13 It was shown that CD105 / CD3Nb-Lipo mediated T cell inhibition of tumor growth in vivo in tumor-bearing mice.

[0036] Fig.14 It showed that CD105 / CD3Nb-Lipo-mediated T cells effectively prolonged the survival time of tumor-bearing mice.

[0037] Fig.15 Schematic diagram showing the synthesis of CD105 / CD3Nb-Lipo ( Fig.15 A) and schematic diagram of the mechanism of action ( Fig.15 B). DETAILED DESCRIPTION

[0038] In a first aspect, the present invention provides a method for preparing a liposome-based T cell agonist based on a nanobody, wherein the method comprises the following steps:

[0039] (1) dissolving distearoylphosphatidylcholine, cholesterol succinate monoester, and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent to prepare a pretreated product;

[0040] (2) using a microporous filter membrane to push the pretreated product through a pusher to obtain nanoliposomes;

[0041] (3) allowing the nanobody and DSPE-PEG-NHS to undergo an addition reaction to prepare nanobody micelles;

[0042] (4) adding Lipo to the nanobody micelles and incubating them to prepare a nanobody-based liposome T cell agonist.

[0043] The nano-antibody-based liposome T cell agonist of the present invention is relatively simple and convenient to prepare and operate, easy to mass produce, and easy to modify and load drugs, and easy to integrate multifunctionality and combined therapy, and is therefore an ideal T cell agonist.

[0044] The immunoliposomes used in the present invention are modified with molecules having specific immune functions, such as antibodies. These functional modifications can endow the liposomes with the ability to recognize cell antigen molecules, and integrate the characteristics of simple liposome preparation, good biocompatibility, weak immunogenicity, and non-toxicity with the antigen-specific targeting of antibodies, and can be widely used in drug-carrying preparations.

[0045] In the present invention, nanoantibodies such as CD3Nb and nanoantibodies specifically targeting tumors such as CD105Nb are modified on immunoliposomes to design bispecific immunoliposomes that have the functions of activating T lymphocytes and targeting tumor sites, promoting the local enrichment of cytotoxic T lymphocytes in the tumor and exerting an effective anti-tumor effect.

[0046] Compared with traditional antibodies, the nano antibodies used in the present invention have simple preparation processes, low immunogenicity, good thermal stability, small molecular weight, strong penetrability, easy clearance in the body, high targeting specificity, and better expression, water solubility and stability. Therefore, the bispecific immunoliposomes prepared by the present invention using nano antibodies as structural units can utilize the many excellent properties of nano antibodies, so that the immunoliposomes have a smaller volume and stability. As a "vehicle" for T cell therapy, it can not only give T cells highly specific tumor targeting, but also may enhance the ability of T cells to infiltrate into relatively dense tissues around tumors and inside tumor entities, which is helpful for promoting T cells to kill tumor cells and enhance the anti-tumor treatment effect.

[0047] The PEGylated liposomes used in the present invention can increase the stability of the liposomes in plasma, reduce the binding and destruction of the liposomes by opsonins, plasma proteins and cells in the plasma, and can effectively avoid being recognized by phagocytes of the mononuclear phagocyte system of organs such as the liver and spleen, thereby significantly prolonging their circulation time.

[0048] The method of the present invention uses PEGylated liposomes as carriers of nanoantibodies to construct liposome-based T cell agonists based on nanoantibodies, so as to complement the respective functions and advantages of PEGylated liposomes and tumor-specific / T cell-specific nanoantibodies, thereby more effectively and lastingly mediating the specific anti-tumor effect of T cells killing tumor cells, and is a new and more efficient method for targeted tumor treatment.

[0049] In addition, in some preferred embodiments of the present invention, CD105Nb is used. Membrane antigen CD105 is commonly found in tumor interstitial blood vessels (tumor angiogenesis is one of the most closely related factors affecting tumor growth rate), and is related to vascular endothelial cell proliferation, also known as endothelin (Endoglin, ENG), which is highly expressed in neovascular endothelial cells in tumor interstitium and is considered to be one of the highly specific marker molecules in tumor neovascular endothelial cells. These preferred embodiments of the present invention are the first to target CD105 and simultaneously couple the immunoliposome T cell agonist of CD3 antibody. Further targeting the CD105 antigen that is generally highly expressed in solid tumor TME as an anti-tumor treatment target, the liposome T cell agonist based on nano antibodies has positive strategic significance in the field of exploring new methods for tumor diagnosis and treatment. In order to enhance the targeting and efficient T cell tumor killing effect based on T cell immune anti-tumor therapy, the present invention uses CD105 as a specific target for anti-tumor immunotherapy in some preferred embodiments, and conceives, designs and constructs this liposome T cell agonist based on nano antibodies (which can be named CD105 / CD3Nb-Lipo herein). The agonist can simultaneously bind with high affinity and high specificity to the differentiation antigen CD3 antigen on the surface of T cells and the CD105 antigen highly expressed in the tumor microenvironment (especially the tumor vascular endothelium), thereby forming an immune synapse and signal connection between T cells and tumor cells that effectively activates T cells, thereby achieving direct killing of tumor cells.

[0050] The synthesis diagram of the agonist of the present invention can be found in Fig.15 A.

[0051] Preferably, in step (1), the pre-treated product can be prepared by thin film hydration and ultrasonic film stripping. In the thin film hydration method, after the organic solvent is removed by a rotary evaporator, the liposome forms a liposome film in the rotary evaporation bottle, and then the film is fully oscillated with the water solvent for hydration. In the ultrasonic film stripping method, the hydrated liposome film in the rotary evaporation bottle is ultrasonically treated by an ultrasonic instrument, so that the hydrated liposome film falls off the rotary evaporation bottle.

[0052] Preferably, in step (2), the squeezer is a liposome squeezer.

[0053] Preferably, in step (3), the addition reaction utilizes the amino group on the nanobody and the maleimide group on the nanoliposome to cause the nanobody and the nanoliposome to undergo an addition reaction.

[0054] Preferably, in step (4), the nanobody is modified on the surface of the nanoliposome by a post-insertion method, and then purified to obtain the nanobody-based liposome T cell agonist of the present invention. The post-insertion method utilizes the amino group of the nanobody molecule and the maleimide group contained in distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)-succinimide ester (NHS) (sometimes referred to as DSPE-PEG-NHS in this article, and the raw material can be purchased from Aladdin (Aladdin), China) to carry out a chemical cross-linking reaction to generate nanobody micelles. The DSPE end of the nanobody micelle is lipophilic, and the DSPE end can be effectively inserted into the liposome molecule, while the nanobody at the other end is modified on the liposome surface. In DSPE-PEG-NHS, (the PEG part thereof can be polyethylene glycol 1000 to 3000, for example, PEG 1000, PEG2000 or PEG3000).

[0055] In some preferred embodiments, the molar ratio of distearoylphosphatidylcholine:cholesterol succinate:distearoylphosphatidylethanolamine-polyethylene glycol (wherein the polyethylene glycol (PEG) can be polyethylene glycol 1000 to 3000, for example, PEG 1000, PEG2000 or PEG3000) is 3:2:(0.005-0.015), for example, 3:2:0.005, 3:2:0.010 or 3:2:0.015, preferably 3:2:0.015.

[0056] In other preferred embodiments, the organic solvent is selected from at least one of chloroform, methanol, ethanol, dichloromethane and acetone. More preferably, the organic solvent is a mixed solvent of chloroform and methanol. Further preferably, the volume ratio of chloroform to methanol in the mixed solvent is 1:4 to 4:1, for example, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1 or 4:1, preferably 4:1.

[0057] In other preferred embodiments, the pore size of the microporous filter membrane is 50 nm to 200 nm, for example, 200 nm, 100 nm or 50 nm, preferably 100 nm. The microporous filter membrane can be selected from one of a polycarbonate membrane, a polyethersulfone membrane, a cellulose membrane and a nylon membrane.

[0058] In other preferred embodiments, the pushing is performed 3 to 20 times, for example, 5 times, 10 times, 15 times or 20 times, preferably 8 to 10 times.

[0059] In other preferred embodiments, the nanobodies include T cell-targeted nanobodies and tumor-targeted nanobodies, and the tumor-targeted nanobodies are nanobodies that target surface-specific markers of tumor cells or tumor microenvironment cells.

[0060] In other preferred embodiments, the T cell targeting nanobody is a CD3 nanobody targeting T cells.

[0061] In other preferred embodiments, the tumor-targeting nanobody is selected from at least one of the nanobodies targeting tumor neovascular endothelial cells CD105 (sometimes referred to herein as CD105Nb), anti-fibroblast activation protein nanobody (sometimes referred to herein as Anti-FAP Nb), and anti-human epidermal receptor protein nanobody (sometimes referred to herein as Anti-Her Nb).

[0062] In other particularly preferred embodiments, the nanobody is a combination of an anti-CD3 nanobody (abbreviated as CD3Nb) and an anti-CD105 nanobody (abbreviated as CD105Nb). The CD3Nb targets the T cell CD3 epitope, while the CD105Nb targets the tumor neovascular endothelial cell CD105 antigen. Preferably, the CD3Nb comprises CD3VHH and a label protein HIS-tag, and / or the CD105Nb comprises CD105VHH and a label protein HIS-tag. In some more preferred embodiments, the amino acid sequence of the CD105Nb is as shown in SEQ ID NO.1, and the nucleotide sequence encoding the CD105Nb may be as shown in SEQID NO.2; and / or the amino acid sequence of the CD3Nb is as shown in SEQ ID NO.3, and the nucleotide sequence encoding the CD3Nb may be as shown in SEQ ID NO.2.

[0063] In some more specific embodiments, the method may include: constructing expression systems for CD3Nb and CD105Nb respectively, purifying and preparing CD3Nb and CD105Nb respectively by in vitro induction and amplification culture; preparing nanoliposome Lipo by thin film hydration method; mixing CD3Nb, CD105Nb and DSPE-PEG-NHS, using the amino group on the nanoantibody to react with the maleimide group on the DSPE-PEG-NHS to form DSPE-PEG Nb micelles, and modifying the nanoantibody on the DSPE-PEG Nb on the Lipo surface by the post-insertion method to prepare the nanoantibody-based liposome T cell agonist CD105 / CD3Nb-Lipo.

[0064] In other preferred embodiments, the nanobody is prepared by the following method: (i) transforming Escherichia coli with a recombinant plasmid containing a nucleotide sequence encoding the nanobody to obtain a nanobody-expressing strain; (ii) amplifying and culturing the nanobody-expressing strain and inducing the expression of the nanobody with IPTG to obtain a nanobody culture; (iii) purifying the nanobody from the nanobody culture.

[0065] In other preferred embodiments, the temperature of the amplification culture is 16-37°C, for example, 20°C, 25°C, 28°C, 30°C or 35°C, preferably 28°C.

[0066] In other preferred embodiments, the concentration of IPTG used to induce expression is 0.1-2 mmol / L, for example, 0.1 mmol / L, 0.2 mmol / L, 0.5 mmol / L, 0.8 mmol / L, 1.0 mmol / L, 1.2 mmol / L, 1.5 mmol / L, 1.8 mmol / L or 2.0 mmol / L, preferably 1 mmol / L.

[0067] In a second aspect, the present invention provides a liposome T cell agonist based on nanoantibodies, wherein the agonist is prepared by the method described in the first aspect of the present invention.

[0068] The agonist of the present invention is generally a nanoliposome particle. In some preferred embodiments, the particle size distribution of the agonist is 90-110 nm, for example, 100 nm.

[0069] In other preferred embodiments, the encapsulation rate of the agonist is above 90%, for example, above 90%, above 91%, above 92%, above 93%, above 94%, above 95% or above 96%.

[0070] The inventors have found through experiments that the nano-antibody-based liposome T cell agonist of the present invention has the following effects: it can simultaneously target and specifically bind to T cells and tumor cells, mediate T cell activation, stimulate T cells to significantly proliferate, upregulate the expression of surface activation molecule markers of T cells (such as CD25 and CD69), and promote the secretion of factors such as IFN-γ, TNF-α, IL-2, etc. by T cells, and can effectively inhibit tumor cell growth, reduce tumor volume, and prolong survival in vivo and in vitro.

[0071] In a third aspect, the present invention provides the use of the nano-antibody-based liposome T cell agonist described in the second aspect of the present invention in the preparation of an anti-tumor drug, wherein the drug is used in the following aspects: (1) specifically targeting and binding to specific markers on the surface of T cells, tumor cells and / or tumor microenvironment cells; (2) activating T cells to exert anti-tumor effects.

[0072] In other preferred embodiments, the surface specific marker is selected from at least one of T cell surface CD3 protein, tumor neovascular endothelial cell CD105 protein, tumor fibroblast activation FAP protein and human epidermal receptor Her protein.

[0073] In other preferred embodiments, the activated T cells exerting anti-tumor effects include activating T cells to proliferate, activate and / or secrete inflammatory cytokines.

[0074] In other preferred embodiments, the tumor is selected from liver cancer, breast cancer, melanoma or lung cancer. Accordingly, the tumor cell can be a liver cancer cell, a breast cancer cell, a melanoma cell or a lung cancer cell.

[0075] The schematic diagram of the mechanism of action of the agonist of the present invention can be found in Fig.15 B.

[0076] The present invention is further described in detail below in conjunction with examples. The examples given are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0077] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0078] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0079] Example 1 Preparation and Characterization of CD105 / CD3Nb-Lipo

[0080] In this embodiment, the preparation method of CD105 / CD3Nb-Lipo comprises the following steps:

[0081] (1) Prokaryotic induction of CD105Nb and CD3Nb expression

[0082] The CD105Nb recombinant plasmid (the amino acid sequence of CD105Nb is shown in SEQ ID NO.1, and the nucleotide sequence of CD105Nb is shown in SEQ ID NO.2) and the CD3Nb recombinant plasmid (the amino acid sequence of CD3Nb is shown in SEQ ID NO.3, and the nucleotide sequence of CD3Nb is shown in SEQ ID NO.4) were transformed into Escherichia coli BL21 (DE3), respectively. The IPTG induction conditions including the induction concentration, induction time and induction temperature of IPTG were optimized, and finally the optimal conditions for large-scale prokaryotic expression of CD105Nb and CD3Nb were determined to be: IPTG concentration of 0.1mM, temperature of 16°C, and induction time of 6h. The bacterial supernatant after lysis was purified using the BeyoGoldTMHis-tag purification kit (Beyotime, China) to obtain crude extracts of CD105Nb and CD3Nb, respectively. The crude extracts were further centrifuged and purified using a 3kDa ultrafiltration centrifuge tube to obtain purified CD105Nb and CD3Nb. The protein concentration of the purified solution was detected using a protein quantification kit (Beyotime, China), and different amounts of dd H2O (double distilled water) were added to adjust each purified solution to a CD105Nb and CD3Nb aqueous solution of each target concentration.

[0083] Table 1. Amino acid and nucleotide sequences of CD105Nb and CD3Nb.

[0084]

[0085]

[0086] (2) Preparation of Nanoliposomes

[0087] Weigh each reagent in a ratio of 3:2:0.015 (i.e. DSPC:Chol:DSPE-PEG-2000) of distearoylphosphatidylcholine (DSPC):cholesterol succinate monoester (Chol):distearoylphosphatidylethanolamine-polyethylene glycol 2000 (i.e. DSPE-PEG-2000). After adding chloroform / methanol (4:1, v / v) mixed solvent for ultrasonic dissolution, the chloroform / methanol mixed solvent was completely evaporated to dryness under vacuum at 50°C by a rotary evaporator. Add 1×PBS buffer to obtain a lipid solution. Ultrasonic demembraning of the lipid solution was performed by a cell ultrasonic disruptor in an ice bath (power 30%, interval 3s, operation 3s). The ultrasonically dispersed lipid solution was loaded into a liposome squeezer and squeezed back and forth 10 times using a polycarbonate membrane with a pore size of 100 nm to obtain nanoliposomes (Liposome, Lipo), which were stored in a refrigerator at 4°C.

[0088] (3) Preparation of CD105 / CD3Nb-Lipo

[0089] The CD105 nanoantibody aqueous solution and the CD3 nanoantibody aqueous solution prepared in step (1) were dissolved in 1×PBS (0.01M phosphate concentration) buffer (pH 7.4) and mixed in a (1:1) equimolar ratio to obtain a nanoantibody mixed solution. DSPE-PEG2000-NHS (purchased from Aladdin, China) was measured at a CD105Nb:CD3Nb:DSPE-PEG2000-NHS molar ratio of 1:1:10, and DSPE-PEG-NHS was dissolved in chloroform in a round-bottom flask. The chloroform solvent was completely evaporated under vacuum conditions at 55°C by a rotary evaporator, and 1×PBS was added to the round-bottom flask for ultrasonic demolding. The CD105 nanoantibody and CD3 nanoantibody mixed solution was added to the solution after ultrasonic demolding. After nitrogen was introduced for 10 minutes, the mixture was sealed, protected from light, and placed in a microplate constant temperature oscillator at a speed of 250 rpm and a temperature of 4°C for 24 hours to obtain nano-antibody micelles (DSPE-PEG Nb micelles). The nano-antibody micelles were added to Lipo, and nitrogen was introduced for 10 minutes, and the mixture was sealed, protected from light, and placed in a microplate constant temperature oscillator at a speed of 250 rpm / min and a temperature of 4°C for slow shaking and incubation for 12 hours. Finally, free unbound antibodies were removed through a Sephadex G-50 dextran gel column to obtain nano-immunoliposomes (CD105 / CD3Nb-Lipo).

[0090] (4) Analysis of the physicochemical properties of CD105 / CD3Nb-Lipo liposome T cell agonists

[0091] The molecular weight and purity of CD105 nanobody and CD3 nanobody were detected by SDS-PAGE electrophoresis.

[0092] Transmission electron microscopy, Malvern particle size analyzer, and UV-visible absorption spectroscopy were used to detect the physical and chemical properties of CD105 / CD3Nb-Lipo liposome T cell agonist, such as particle size, potential, dispersion coefficient, and stability.

[0093] Figure 1 The purification and characterization of nanobodies are shown. Figure 1 The SDS-PAGE electrophoresis results showed that there was no obvious band in the 5th washing liquid lane (W5), indicating that the non-target protein that was not bound to the gel had been eluted cleanly, while in the elution liquid lanes (E1-E5), there was a relatively obvious single band at around 17kDa, which was consistent with the target protein CD105 nanobody ( Figure 1 B) and CD3 nanobody ( Figure 1 A) The relative molecular mass is consistent with the size, and its purity can reach more than 98%.

[0094] Figure 2Shows Lipo( Figure 2 A) CD105Nb-Lipo( Figure 2 B) CD3Nb-Lipo( Figure 2 C)CD105 / CDD3Nb-Lipo( Figure 2 D) The particle size distribution, potential and PDI value of nanoliposomes. Figure 2 As shown, the hydrated particle size of each nanoliposome is about 100nm, and the PDI value is less than 0.2, indicating that the size distribution of the nanoliposome is concentrated and uniform, and the dispersion is good. The prepared nanoliposome materials are all negative, with a Zeta potential of about -40mV, and are all anionic liposomes with good stability.

[0095] The particle size, potential, PDI value and leakage rate of CD105 / CD3Nb-Lipo were measured by Malvern particle size analyzer when the particles were stored in 1×PBS solution at 4℃ and 1×PBS solution at 25℃ in the dark. Figure 3 As shown, the particle size, potential and PDI value of CD105 / CD3Nb-Lipo did not change significantly in 1×PBS solution stored at 4°C (within 30 days), and the stability was good.

[0096] Example 2 Cytotoxicity Experiment of CD105 / CD3Nb-Lipo Nanomaterials

[0097] Human normal liver cells LO2 in the logarithmic growth phase were digested and collected, and then spread on a 96-well cell culture plate after being blown evenly. They were placed in a cell culture incubator until the cells were stable and attached to the bottom of the bottle. RPMI-1640 complete culture medium was used to set 6 concentrations of CD105 / CD3Nb-Lipo at 5μg / mL, 10μg / mL, 20μg / mL, 40μg / mL, and 80μg / mL, and 3 replicates were set for each of the above concentrations. Cultured in a cell culture incubator for 24h or 48h. After the culture, CCK-8 reagent was used to detect the toxic effects of CD105 / CD3Nb-Lipo nanomaterials on the two cells cultured at different culture times. The results are shown in Figure 4 As shown, within the concentration range of 0 to 80 μg / mL, after CD105 / CD3Nb-Lipo was co-incubated with LO2 cells for 24 h and 48 h, the proportion of live cells was close to 100%, proving that CD105 / CD3Nb-Lipo had no toxic effect on normal human cells.

[0098] Example 3 Binding of CD105 / CD3Nb-Lipo to target cells

[0099] CD105 / CD3Nb-Lipo and control group materials were added to 106 After incubation on ice for 30 min, cells were centrifuged, His-tag mAb FITC flow cytometry antibody was added, and incubation continued on ice in the dark for 30 min. Fluorescence detection was performed using a flow cytometer. Figure 5 As shown, it indicates that CD105 / CD3Nb-Lipo has a high binding efficiency with T cells and Bel7404 target cells that highly express CD105.

[0100] Example 4 Effect of CD105 / CD3Nb-Lipo on T cell proliferation

[0101] Peripheral blood mononuclear cells (PBMC) were sorted and collected by flow cytometry, washed with 1×PBS to remove serum, and then 1μL 5mmol / L CFSE (carboxyfluorescein diacetate succinimidyl lipid) solution was added. After incubation in the dark for 10 minutes, the cells were washed three times with RPMI-1640 medium containing 5% serum. Resuspended with 1×PBS, fixed with 4% paraformaldehyde, and stored in the dark at 4°C as the proliferative mother. Bel7404 cells in the logarithmic growth phase were added with 2mL of 25μg / mL mitomycin C solution, incubated for 30 minutes, and washed 5 times with 1×PBS to ensure that there was no residual mitomycin C. After the target cells and each group of materials were co-incubated in a cell culture incubator for 1 hour, CFSE-stained T cells were added at an effect-target ratio of 1:1 for co-incubation. Finally, CD3 + The CFSE fluorescence intensity of T cells was detected by flow cytometry. Figure 6 As shown, under the stimulation of target cells, the proliferation ratio of T cells in the CD105 / CD3Nb-Lipo group was significantly higher than that in the other material groups.

[0102] Example 5 Effect of CD105 / CD3Nb-Lipo on the expression of T cell activation markers CD25 and CD69

[0103] Bel7404 cells treated with mitomycin C (treatment method see Example 4) were digested with trypsin, and sorted T cells were added at an effector-target ratio of 1:1 according to the grouping (Lipo, CD3Nb-Lipo, CD105-Lipo, CD105 / CD3Nb-Lipo), and co-incubated and cultured for 3 days. T cells were collected, 2 μL of flow cytometry antibody Anti-human CD25-PE or Anti-human CD69-PE was added in dark, and detected by flow cytometry. The experimental results are shown in Figure 7 (CD25) and Figure 8As shown in (CD69), under the stimulation of target cells Bel7404 and CD105 / CD3Nb-Lipo, the expression levels of T cell surface activation molecules CD25 and CD69 were significantly higher than those in other material groups, indicating that CD105 / CD3Nb-Lipo can promote T cell activation mediated by Bel7404 target cells.

[0104] Example 6 Effect of CD105 / CD3Nb-Lipo on the secretion of IFN-γ, IL-2, and TNF-α by T cells

[0105] According to the grouping (same as Example 5), relevant materials (Lipo, CD3Nb-Lipo, CD105-Lipo, CD105 / CD3Nb-Lipo) were added to the target cells respectively, and mixed with T cells at a ratio of 1:1 to target, and cultured for 24 hours, and then the supernatant was collected. The ELISA detection kit (Hangzhou Lianke, China) was taken out of the 4°C refrigerator, and the IFN-γ, IL-2, and TNF-α standard solutions were prepared and diluted according to the instructions of the kit. The diluted supernatant and various detection antibodies were added in strict accordance with the operating procedures of the kit instructions, and incubation and plate washing were performed. After terminating the reaction, the OD value of each well in the well plate was detected at a wavelength of 450nm using a full-wavelength microplate reader. The experimental results are as follows. Fig. 9 (IFN-γ), Fig.10 (IL-2) and Fig.11 As shown in (TNF-α), the IFN-γ, TNF-α, and IL-2 secreted by T cells in the CD105 / CD3Nb-Lipo group were significantly higher than those in the control group.

[0106] Example 7: In vitro killing effect of CD105 / CD3Nb-Lipo on target cells

[0107] After Bel7404 target cells were resuspended in complete RPMI-1640 medium, 3 replicate wells were set up in each group. According to the grouping, relevant materials (Lipo, CD3Nb-Lipo, CD105-Lipo, CD105 / CD3Nb-Lipo) were added to the target cells, and T cells sorted by cell flow cytometry were added to the target cells at effector-target ratios of 1:1, 5:1, and 10:1 using complete RPMI-1640 medium.

[0108] After the incubation time is over, the target cells are digested with EDTA-free trypsin to collect the target cells. The apoptosis of the target cells is determined using the AnnexinV-APC / 7-AAD apoptosis detection kit (BioLab, China): 300 μL AnnexinV binding buffer is added to each tube of target cells, followed by 2.5 μL AnnexinV-APC and 2.5 μL 7-AAD, respectively. After incubation on ice in the dark for 30 minutes, the cells are detected by flow cytometry within 1 hour. The experimental results are shown in Figure 2. Fig.12 As shown, the CD105 / CD3Nb-Lipo group had the strongest killing efficiency on Bel7404 target cells, and the killing efficiency increased with the increase of the effector-target ratio.

[0109] Example 8 Treatment of NOD / SCID Mouse Model Bearing Human Xenograft Tumor with CD105 / CD3Nb-Lipo

[0110] When the subcutaneous tumor volume of tumor-bearing mice is about 80-100mm 3 When the tumor volume was approximately equal, mice with roughly equal tumor size were randomly divided into groups. Treatment began on day 0, and was carried out according to the grouping and treatment measures. CD105 / CD3Nb-Lipo and T cell injections were performed through the tail vein of mice on days 0, 7, 14 and 21, respectively. A total of 4 treatments were performed, and the treatment ended one week after the fourth treatment, that is, on the 28th day. From the date of treatment, the tumor volume and body weight of the mice were measured every 4 days and recorded. After the treatment, blood was collected from the mouse eyeballs, the tumor was removed, the tumor weight was weighed, the tumor inhibition rate was calculated, and the mouse tumor volume growth curve was drawn based on the records. The experimental results are as follows Fig.13 and 14 As shown, the tumor volume of each group of mice continued to increase, and CD105 / CD3Nb-Lipo and T cells could significantly inhibit the growth of Bel7404 tumors and prolong the survival time of NOD / SCID mice bearing human liver cancer Bel7404.

Claims

1. A method for preparing a liposome-based T cell agonist based on nanobodies, characterized in that: The method comprises the following steps: (1) dissolving distearoylphosphatidylcholine, cholesterol succinate monoester, and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent to prepare a pretreated product; (2) using a microporous filter membrane to push the pretreated product through a pusher to obtain nanoliposomes; (3) allowing the nanobody and distearoyl phosphatidylethanolamine-polyethylene glycol-succinimide ester to undergo an addition reaction to prepare nanobody micelles; (4) adding the nanoliposome to the nanoantibody micelle for incubation to obtain a nanoantibody-based liposome T cell agonist.

2. The method according to claim 1, characterized in that: The molar ratio of distearoylphosphatidylcholine:cholesterol succinate:distearoylphosphatidylethanolamine-polyethylene glycol is 3:2:(0.005-0.015), preferably 3:2:0.015; and / or The volume ratio of chloroform to methanol in the mixed solvent is 1:4 to 4:1, preferably 4:

1.

3. The method according to claim 1 or 2, characterized in that: The pore size of the microporous filter membrane is 50nm to 200nm, preferably 100nm; The microporous membrane is selected from one of a polycarbonate membrane, a polyethersulfone membrane, a cellulose membrane and a nylon membrane; and / or The pushing is performed 3 to 20 times, preferably 8 to 10 times.

4. The method according to any one of claims 1 to 3, characterized in that: The nanobodies include T cell-targeted nanobodies and tumor-targeted nanobodies. The tumor-targeted nanobodies are nanobodies that target specific markers on the surface of tumor cells or tumor microenvironment cells. Preferably, the T cell targeting nanobody is a CD3 nanobody targeting T cells; In addition, preferably, the tumor-targeting nanobody is selected from at least one of the nanobodies targeting tumor neovascular endothelial cells CD105, nanobodies against fibroblast activation protein FAP, and nanobodies against human epidermal receptor protein Her; It is further preferred that the nanobody is a combination of an anti-CD3 nanobody and an anti-CD105 nanobody.

5. The method according to any one of claims 1 to 3, characterized in that: The nanobody is prepared by the following method: (i) transforming Escherichia coli with a recombinant plasmid containing the Nanobody coding sequence to obtain a Nanobody-expressing strain; (ii) amplifying and culturing the nanobody-expressing strain, and inducing the expression of the nanobody with IPTG to obtain a nanobody culture; (iii) purifying the Nanobody from the Nanobody culture.

6. The method according to claim 5, characterized in that: The temperature of the amplification culture is 16-37°C, preferably 28°C; The concentration of IPTG used to induce expression is 0.1-2 mmol / L, preferably 1 mmol / L.

7. A liposome T cell agonist based on nanoantibodies, characterized in that: The agonist is prepared by the method according to any one of claims 1 to 6.

8. The agonist according to claim 8, characterized in that: The particle size distribution of the agonist is 90-110 nm.

9. Use of the nanobody-based liposome T cell agonist according to claim 7 or 8 in the preparation of an anti-tumor drug, wherein the drug is used in the following aspects: (1) Specific binding to surface markers of targeted T cells, tumor cells, and / or tumor microenvironment cells; (2) Activate T cells to exert anti-tumor effects.

10. The use according to claim 9, characterized in that: The surface specific marker is selected from at least one of T cell surface CD3 protein, tumor neovascular endothelial cell CD105 protein, tumor fibroblast activation FAP protein and human epidermal receptor Her protein; The activated T cells exerting anti-tumor effects include activating T cells to proliferate, activate and / or secrete inflammatory cytokines; and / or The tumor is selected from liver cancer, breast cancer, melanoma or lung cancer.

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