Tobezumab / lanthanide series metal / nucleic acid nano-composite, preparation method and application

Abstract

The invention relates to the technical field of nano-drugs, in particular to a tolbuzumab / lanthanide series metal / nucleic acid nano-composite, a preparation method and application. The mass ratio of the tolbuzumab to the lanthanide series metal to the nucleic acid in the nano-composite is (0.5-5.1): 1: (10-11). The nano compound disclosed by the invention can be used for promoting the activation of a cGAS-STING-STAT1 pathway and blocking an IL6 / IL6R-STAT3 signal pathway at the same time, so that chronic inflammation caused by a CIN phenomenon of tumor cells is converted into anti-tumor immune response.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, specifically to tocilizumab / lanthanide metal / nucleic acid nanocomposites, their preparation methods, and applications. Background Technology

[0002] Chromosomal instability (CIN) frequently occurs during tumor cell growth and is a hallmark of tumors, caused by persistent misseparation of chromosomes during the later stages of mitosis. CIN promotes cancer progression and leads to tumor metastasis. Specifically, on the one hand, CIN can regulate chromosome copy number by increasing the copy number of chromosomes carrying oncogenes and decreasing the copy number of chromosomes carrying tumor suppressor genes, thereby enhancing the adaptability of tumor cells. On the other hand, after CIN occurs, the misseparation of chromosomes results in the formation of "micronuclei." When these micronuclei rupture, they leak genomic DNA into the cytoplasm. This leads to the activation of the cGAS-STING (circular GMP-AMP synthase stimulator of interferon genes) cytoplasmic DNA-sensitive pathway and the downstream atypical NF-κB signaling pathway. Because the formation and rupture of micronuclei continue to occur during tumor progression, they contribute to chronic inflammation through the activation of the STING pathway. This chronic inflammation promotes cell invasion and metastasis. By disrupting the epithelial cells' lethal response to cytoplasmic DNA, tumor cells that develop CIN will use the chronic inflammation caused by the innate immune response to spread to distant organs.

[0003] Recent research indicates that in tumor cells developing CIN, on the one hand, cGAS-STING activates STAT1 through type I interferon, further mediating tumor cell apoptosis and the body's immune system's surveillance of them; on the other hand, activation of the cGAS-STING signaling pathway triggers IL6-STAT3-mediated signaling, which, through the cGAS-STING and atypical NF-κB pathways, leads to inflammatory tolerance in tumor cells, ultimately resulting in tumor cell metastasis. Figure 1 Selective blocking of the IL6 signaling pathway could help drive chronically inflammatory CIN cells toward tumor cell apoptosis and immune surveillance, ultimately leading to an anti-tumor immune response. In TNBC, the cGAS-IL-6-IL-6R signaling pathway is often activated, which is one reason why triple-negative breast cancer is prone to recurrence and metastasis.

[0004] In existing research, the journal *Lanthanide-Nucleotide Coordination Nanoparticles for STING Activation* (Zichao Luo, Xiuqi Liang, Tao He, et al., *Journal of the American Chemical Society*, 2022) disclosed a novel class of STING pathway agonists, Ln-GAMP-NPs, based on lanthanide (Ln) coordinated nanostructures. The study found that Eu-GAMP-NPs nanoparticles promote the activation of type I interferon and the classical NF-κB pathway by activating STING, mediating a systemic anti-tumor immune response. Furthermore, Ln-GAMP-NPs exhibit acid responsiveness. Compared to conventional 2'3'-cGAMP, Ln-GAMP-NPs offer advantages such as a simpler synthesis scheme, higher stability, easier cellular uptake, and the ability to elicit a stronger systemic immune response. However, Ln-GAMP-NPs cannot block the IL6-STAT3 signaling pathway in tumor cells, have no effect on chronic inflammation caused by tumor cells, and have limited therapeutic efficacy against tumor cell metastasis. Furthermore, current technologies do not offer nanomedicines designed from the perspective of reversing chronic inflammation in tumors to create an anti-tumor immune response. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a tocilizumab / lanthanide metal / nucleic acid nanocomposite, its preparation method, and its applications. This invention creatively transforms chronic tumor inflammation into an anti-tumor immune response by designing a tocilizumab / lanthanide metal / nucleic acid nanocomposite formed by one-step encapsulation of the IL-6R antibody blocker tocilizumab within a lanthanide metal / nucleic acid nanocomposite. This nanocomposite can promote the activation of the cGAS-STING-STAT1 pathway while blocking the IL6 / IL6R-STAT3 signaling pathway, thereby converting chronic inflammation caused by CIN in tumor cells into an anti-tumor immune response.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a tocilizumab / lanthanide metal / nucleic acid nanocomposite, wherein the mass ratio of tocilizumab, lanthanide metal, and nucleic acid is (0.5~5.1):1:(10~11).

[0007] Preferably, the lanthanide metal is selected from any one or more of lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), samarium (Sm), europium (Eu), gadolinium (Gd), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), and yttrium (Y).

[0008] Preferably, the nucleic acid is 5'-guanosine monophosphate (GMP) and 5'-adenosine monophosphate (AMP).

[0009] Preferably, the mass ratio of 5'-guanosine monophosphate (GMP) to 5'-adenosine monophosphate (AMP) is 1.15:1.

[0010] Preferably, the mass ratio of tocilizumab, lanthanide metal, and nucleic acid is (0.5~5.05):1:11. Exemplarily, the mass ratio of tocilizumab, lanthanide metal, and nucleic acid is any one of 0.5:1:11, 1.26:1:11, 2.5:1:11, or 5.05:1:11, or a value between two of these.

[0011] Preferably, the particle size of the nanocomposite is 40-50 nm.

[0012] Secondly, the present invention provides a method for preparing the tocilizumab / lanthanide metal / nucleic acid nanocomposite, comprising the following steps: Tocilizumab was added to a nucleic acid solution, stirred and mixed, and then a lanthanide metal ion solution was added. After the reaction solution produced the Tyndall effect, the reaction was continued for a period of time, centrifuged, and the supernatant was discarded to obtain the nanocomposite.

[0013] Preferably, the solvent in the nucleic acid solution and the lanthanide metal ion solution is a buffer solution with a pH of 7-8.

[0014] Preferably, the buffer solution is composed of 4-hydroxyethylpiperazine ethanesulfonic acid.

[0015] Thirdly, the present invention provides the application of the tocilizumab / lanthanide metal / nucleic acid nanocomposite or the tocilizumab / lanthanide metal / nucleic acid nanocomposite prepared by the preparation method in the preparation of antitumor drugs.

[0016] Preferably, the antitumor drug is a drug that promotes the activation of the cGAS-STING-STAT1 pathway and blocks the IL6 / IL6R-STAT3 signaling pathway.

[0017] Preferably, the antitumor drug is a drug for treating breast cancer and melanoma in situ and metastasized.

[0018] Preferably, the concentration of the active ingredient in the nanocomposite is tocilizumab ≥ 2.5 μg / mL.

[0019] The beneficial effects of this invention are: The tocilizumab / lanthanide metal / nucleic acid nanocomposite of the present invention can activate the cGAS-STING pathway and mediate a systemic anti-tumor immune response on the one hand, and block the IL6 / IL6R-STAT3 signaling pathway on the other hand, reduce chronic inflammation of tumor cells, and induce tumor cell apoptosis. Ultimately, it transforms the chronic inflammation caused by tumor cell CIN into an anti-tumor immune response, breaks down the chronic inflammation caused by tumor CIN, drives it to the anti-tumor immunotherapy side, and ultimately reduces tumor recurrence and metastasis. Attached Figure Description

[0020] Figure 1 A diagram illustrating the mechanism by which cGAS drives chronic inflammatory responses.

[0021] Figure 2 The diagrams show the preparation and characterization of the tocilizumab / lanthanide metal / nucleic acid nanocomposite (where (ab) is a schematic diagram of nanoparticle preparation, (cd) is a TEM electron microscope image of the nanoparticles (scale bar 100 nm), (ef) is an elemental distribution diagram of the two nanoparticles, (g) is a detection diagram of the encapsulation efficiency of Tcz-Eu-GAMP-NP, and (h) is a detection diagram of the drug loading of Tcz-Eu-GAMP-NP).

[0022] Figure 3 The in vitro evaluation diagram of the biological function of Tcz-Eu-GAMP-NP is shown below (where (a) the effect of tocilizumab on the STING-STAT3 and STING-STAT1 pathways; (b) the effect of GAMP-NP on the STING-STAT1 pathway; (c) the toxicity evaluation of Tcz-Eu-GAMP-NP on tumor cells; (df) the toxicity evaluation of Tcz-Eu-GAMP-NP on immune cells; (gh) the expression of co-stimulatory factors CD86 and CD83 after Tcz-Eu-GAMP-NP treatment of BMDCs; (kl) the expression of co-stimulatory factors CD86 and CD83 in BMDM cells; (ij, mn) the detection of TNF-α and IL-1β in BMDC and BMDM cells; (op) the detection of the presentation effect of BMDC cells on OVA model antigen after different treatments).

[0023] Figure 4In vivo animal evaluation images of Tcz-Eu-GAMP-NPs prepared in Example 1 (wherein, (a) tumor volume growth curve of mouse 4T1 model; (b) tumor volume growth curve of mouse B16F10; (c) survival time of mouse 4T1 model; (d) survival time of mouse B16F10 model; (e) H&E staining images of various tissues in mouse breast cancer model).

[0024] Figure 5 This diagram illustrates the mechanism by which Tcz-Eu-GAMP-NP drives the transformation of chronic inflammation into an anti-tumor immune response in tumor cells. Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical solution of the invention, the invention will be further described in detail below with reference to specific embodiments. Example 1: Preparation of tocilizumab / lanthanide metal / nucleic acid nanocomposite

[0026] The lanthanide metals in this invention are 16 elements excluding the radioactive lanthanide element promethium (Pm), including lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), samarium (Sm), europium (Eu), gadolinium (Gd), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), and yttrium (Y).

[0027] The tocilizumab is abbreviated as Tcz.

[0028] The following uses europium (Eu) as an example to illustrate the preparation process of tocilizumab / lanthanide metal / nucleic acid nanocomposite (Tcz@Eu-GAMP-NPs). Other lanthanide metals can be prepared using the same method to obtain drugs with the same efficacy.

[0029] Preparation process (see) Figure 2 ab) includes the following steps: (1) Prepare a 10mM HEPES (i.e., 4-hydroxyethylpiperazine ethanesulfonic acid) buffer solution in advance, and adjust the pH to 7.4 with NaOH solution.

[0030] (2) Prepare 5mM, 10mM, and 10mM solutions of LnCl3, 5'-guanosine monophosphate (GMP), and 5'-adenosine monophosphate (AMP) using the HEPES buffer described above. Specifically, dissolve 19.084 mg EuCl3·6H2O in 10 mL of HEPES buffer, 46.118 mg GMP in 10 mL of HEPES buffer, and 40.12 mg AMP in 10 mL of HEPES buffer.

[0031] (3) Mix the GMP solution and AMP solution in equal volumes in advance.

[0032] (4) Take 5.45 mL of aqueous solution into a 10 mL vial, then add 500 μL of a mixed solution of GMP and AMP, and stir at 500-600 r / min for 10 minutes.

[0033] (5) Then add 0.25 mg of tocilizumab and continue stirring at 500-600 r / min for 10 minutes.

[0034] (6) Then add 250 μL of EuCl3 to the vial. At this time, the reaction solution is opalescent and Tyndall effect will occur after light exposure.

[0035] (7) Continue the reaction for 15 minutes, then centrifuge at 10000 r / min for 10 minutes.

[0036] (8) Discard the supernatant, then add 5 ml of water and disperse the nanomedicine under 4-degree ultrasonic conditions.

[0037] (9) Continue centrifuging at 10000 r / min for 10 minutes, discard the supernatant to wash the nanomedicine, and then continue to step 8 to obtain the Tcz-Eu-GAMP-NPs nanocomposite. The mass ratio of tocilizumab, Eu, GMP and AMP in the nanocomposite is 250:197.87:1152.95:1003, that is, 1.26:1:5.83:5.07.

[0038] Example 2: Preparation of tocilizumab / lanthanide metal / nucleic acid nanocomposite

[0039] The preparation method is the same as in Example 1, except that: in step (5), the amount of tocilizumab is 0.10 mg; the mass ratio of tocilizumab, Eu, GMP and AMP in the obtained nanocomposite is 100:197.87:1152.95:1003, that is, 0.5:1:5.83:5.07.

[0040] Example 3: Preparation of tocilizumab / lanthanide metal / nucleic acid nanocomposite

[0041] The preparation method is the same as in Example 1, except that: in step (5), the amount of tocilizumab is 0.50 mg; the mass ratio of tocilizumab, Eu, GMP and AMP in the obtained nanocomposite is 500:197.87:1152.95:1003, that is, 2.52:1:5.83:5.07.

[0042] Example 4: Preparation of tocilizumab / lanthanide metal / nucleic acid nanocomposite

[0043] The preparation method is the same as in Example 1, except that: in step (5), the amount of tocilizumab is 1.00 mg; the mass ratio of tocilizumab, Eu, GMP and AMP in the obtained nanocomposite is 1000:197.87:1152.95:1003, that is, 5.05:1:5.83:5.07.

[0044] Comparative Example 1: Preparation of Lanthanide Metal / Nucleic Acid Nanocomposites

[0045] The preparation method is the same as in Example 1, except that tocilizumab was not added.

[0046] Example 1: Characterization of tocilizumab / lanthanide / nucleic acid nanocomposite

[0047] The nanocomposites prepared in Example 1 and Comparative Example 1 were characterized by TEM electron microscopy (scale bar 100 nm), and the results are shown in the figure. Figure 2 As can be seen from TEM, the particle size of Eu-GAMP-NP prepared in Comparative Example 1 and Tcz-Eu-GAMP-NP prepared in Example 1 is 40~50 nm, both of which are nanoscale drug complexes.

[0048] The elemental distribution of nanoparticles before and after drug loading (i.e., Comparative Example 1 and Example 1) was characterized using SEM-EDS scanning. Figure 2 EDS analysis showed that the main elements in the sample were C, O, P, S and Eu, with uniform element distribution, consistent with the expected chemical composition.

[0049] Further evaluation was conducted on the encapsulation efficiency and drug loading of tocilizumab by Eu-GAMP-NP. Figure 2 (gh), found that within the tocilizumab dosage range defined in this invention, Eu-GAMP-NP encapsulates tocilizumab at a rate of over 70%, especially when the tocilizumab mass is 0.1~0.5 mg (Examples 1~3), the encapsulation rate of Eu-GAMP-NP on tocilizumab can reach almost 100%, and the drug loading also increases with the increase of antibody mass.

[0050] Example 2: In vitro evaluation of the biological function of tocilizumab / lanthanide / nucleic acid nanocomposite

[0051] When used in animal and cell experiments, the preparation of nanomedicines must be carried out in a sterile operating table. Except for toluizumab, the required solutions must be sterilized by high-temperature steam, and the reagents must be sterilized by ultraviolet light in advance.

[0052] Further research was conducted using Tcz-Eu-GAMP-NP nanomedicine to explore the scientific hypothesis of driving the transformation of chronic inflammation caused by chromosomal instability in tumor cells into anti-tumor immune surveillance. Western blotting results showed that after treatment with Tcz-Eu-GAMP-NP nanomedicine, tocilizumab at 2.5 μg / mL effectively blocked the expression of STAT3 protein in tumor cells and enhanced the activation of STAT1 protein. Simultaneously, 10 μg / mL of GAMP-NP efficiently enhanced the activation of STAT1 and IRF-3, and enhanced the activation of the cGAS-STING-STAT1 pathway. Figure 3 ab), mediating the death of downstream tumor cells ( Figure 3 c).

[0053] The effect of Tcz-Eu-GAMP-NP nanomedicine on the activity of immune cells was further evaluated using the MTT assay. Figure 3 As shown in d-3f, even at a relatively high concentration (20 μg / mL), Tcz-Eu-GAMP-NP showed no significant toxicity to immune cells. Furthermore, the effects of the Tcz-Eu-GAMP-NP nanomedicine on APC cells were evaluated, revealing that it efficiently activated the maturation and activation of BMDC and BMDM cells, and increased the expression of APC cell co-stimulatory molecules such as CD83 and CD86. Figure 3 Further analysis of cytokines produced by APC cells revealed that the Tcz-Eu-GAMP-NP nanomedicine increased the production of the tumor-killing cytokine TNF-α, but had no significant effect on the pro-inflammatory factor IL-1β. This indicates that the Tcz-Eu-GAMP-NP nanomedicine has the potential value of reducing inflammatory responses and increasing anti-tumor immunity. Figure 3 Simultaneously, the antigen presentation function was evaluated, and it was found that Tcz-Eu-GAMP-NP efficiently promoted the cross-presentation of the model antigen OVA (ij,mn); Figure 3 In summary, the Tcz-Eu-GAMP-NP nanomedicine can block the IL6 / IL6R-STAT3 signaling pathway in tumor cells, promote the activation of the cGAS-STING-STAT1 pathway, and promote the activation and proliferation of antigen-presenting cells.

[0054] Example 3: In vivo animal evaluation of tocilizumab / lanthanide / nucleic acid nanocomposite

[0055] First, mouse subcutaneous breast cancer tumor models and mouse melanoma models were established, with the day of inoculation designated as day 0. When the tumor grew to 100 mm... 3Animals were treated with Tcz-Eu-GAMP-NPs nanomedicine (100 μg / animal), toxolium antibody alone (20 μg / animal), Eu-GAMP-NPs nanomedicine (100 μg / animal), and toxolium antibody + 2,3,-cGAMP (Tcz + cGAMP, 20 μg / animal + 10 μg / animal), with a saline control group. Each group received three doses, with four days between each dose. Figure 4 As shown in ab, the Tcz-Eu-GAMP-NPs nanomedicine group significantly inhibited tumor growth in mice. Figure 4 As can be seen from the CD, the Tcz-Eu-GAMP-NPs nanomedicine group prolonged the survival of mice. Further H&E staining was performed on various tissues in the mouse breast cancer model. Figure 4 As can be seen from the results, obvious metastatic lesions appeared in the lungs of mice in the saline group, while no metastatic lesions were found in any organs in the Tcz-Eu-GAMP-NPs nanomedicine group, indicating that the Tcz-Eu-GAMP-NPs nanomedicine inhibited the metastasis of breast cancer tumors in mice.

[0056] Figure 5 This diagram illustrates the mechanism by which Tcz-Eu-GAMP-NP drives the transformation of chronic inflammation into an anti-tumor immune response in tumor cells. Validation at the cellular level revealed that Tcz-Eu-GAMP-NP responsively releases tocilizumab and Eu-GAMP-NP in colorectal cancer cells. Tocilizumab blocks the IL6 / IL6R-STAT3 signaling pathway, reducing chronic inflammation in tumor cells and leading to tumor cell apoptosis. Simultaneously, Eu-GAMP-NP promotes the activation of the cGAS-STING-STAT1 pathway, synergistically enhancing the activation of the intrinsic endogenous STING-STAT1 pathway in CIN cells, further promoting type I interferon production and enhancing immune killing. Furthermore, Eu-GAMP-NP efficiently promotes the maturation and activation of APCs such as dendritic cells and macrophages, enhancing their recognition and uptake of tumor cells.

[0057] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A tocilizumab / lanthanide metal / nucleic acid nanocomplex, characterized in that, The mass ratio of the tocilizumab, lanthanide metal, and nucleic acid is (0.5-5.1):1:(10-11).

2. The tocilizumab / lanthanide metal / nucleic acid nanocomplex of claim 1, wherein, The lanthanide metal is selected from any one or more of lanthanum, cerium, praseodymium, neodymium, terbium, dysprosium, holmium, erbium, samarium, europium, gadolinium, thulium, ytterbium, lutetium, scandium, and yttrium.

3. The tocilizumab / lanthanide metal / nucleic acid nanocomplex of claim 1, wherein, The nucleic acid is 5'-guanosine monophosphate and 5'-adenosine monophosphate.

4. The tocilizumab / lanthanide metal / nucleic acid nanocomplex of claim 3, wherein, The mass ratio of the 5'-guanosine monophosphate and 5'-adenosine monophosphate is 1.15:

1.

5. The tocilizumab / lanthanide metal / nucleic acid nanocomposite according to any one of claims 1 to 4, characterized in that, The particle size of the nanocomposite is 40-50 nm.

6. A method of preparing the tocilizumab / lanthanide metal / nucleic acid nanocomplex according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: The tocilizumab is added to the nucleic acid solution, and after stirring and mixing, the lanthanide metal ion solution is added. After the obtained reaction solution produces a Tyndall effect, the reaction is continued for a period of time, centrifugation is performed, and the supernatant is discarded to obtain the nanocomposite.

7. Use of the tocilizumab / lanthanide metal / nucleic acid nanocomposite of any one of claims 1-5 or the tocilizumab / lanthanide metal / nucleic acid nanocomposite prepared by the preparation method of claim 6 in the preparation of an antitumor drug.

8. Use according to claim 7, characterized in that, The antitumor drug is a drug that promotes the activation of the cGAS-STING-STAT1 pathway and blocks the IL6 / IL6R-STAT3 signaling pathway.

9. Use according to claim 7, characterized in that, The antitumor drug is a drug for treating in situ and metastatic breast cancer and melanoma.

10. Use according to claim 7, characterized in that, The effective component concentration of the nanocomposite is tocilizumab≥2.5 μg / mL.

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