Synthesis of artificial immunoglobulin and its pharmaceutical applications

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to synthesis of artificial immunoglobulin and application thereof in medicine. LYS and PD-L1 LYS / Plus is obtained through two-step mild self-assembly. The artificial immunoglobulin (IgPbeta) targeting PD-L1 obtained by the application can effectively degrade PD-L1 in cytoplasm, cell membrane and exosome of tumors in vitro and in vivo, while maintaining high tumor specificity and biological safety. The medicine including the substance prepared by the synthesis method of the application is not only effective in degrading tumor PD-L1 and realizing long-term remission of COAD and SKCM, but also avoids immune-related adverse events (IrAES), and further enables us to develop artificial protein medicine for regulating cell membrane and cytoplasm protein content through polypeptide foldamer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the synthesis of artificial immunoglobulin and its application in medicine. Background Art

[0002] Emerging immune checkpoint blockade (ICB) therapies, particularly those targeting programmed cell death receptor 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), have revolutionized the treatment paradigm for cancer, bringing cancer immunotherapy to the forefront. While existing antibodies blocking PD-L1 and PD-1 have shown significant benefit in treating a variety of advanced cancers, over 60% of patients fail to achieve durable remissions, and some even fail to respond to these agents. In these settings, antibody-based therapies block PD-1 or PD-L1 at the cell surface, but their efficacy can be diminished by a series of immune evasion mechanisms involving PD-L1: 1) compensatory expression of PD-L1; 2) endocytosis and subsequent degradation of the antibody; 3) re-accumulation of PD-L1 on recycling endosomes; and 4) neutralization of overexpressed PD-L1 on tumor exosomes by anti-PD-L1 antibodies.

[0003] Therefore, reducing the amount of PD-L1 in tumor cells, including in the cytoplasm, membranes, and exosomes, is a crucial factor in ensuring reliable and durable targeting of PD-L1 as an immunosuppressive molecule. Furthermore, antibody-based ICB therapies are plagued by extremely high costs, demanding storage conditions, high immunogenicity, and acquired resistance due to compensatory effects and / or anti-antibody resistance. To address these issues, several technologies and approaches, such as proteolysis-targeting chimeras (PROTACs), lysosome-targeting chimeras (LYTACs), and molecular glues, have been developed to degrade ICB-associated proteins through diverse molecular properties. While these approaches have achieved some success in overcoming innate / acquired resistance and drug barriers to antibody-based ICB therapies, significant challenges remain: 1) identifying potential beneficiaries of tumor-specific PD-L1 degradation therapy; and 2) accurately and effectively reducing the abundance of PD-L1 in tumor cells.

[0004] To explore potential candidates for tumor PD-L1 degradation therapies, the association between commonly used tumor-targeting molecules and tumor PD-L1 expression can be assessed by analyzing TCGA datasets containing PD-L1-positive clinical samples. Alternatively, biochemical analysis of COAD and SKCM anti-PD-1-resistant PDX mouse models can be performed to assess tumor PD-L1 overexpression and the corresponding CD8+ T cell immunosuppression. This can then be used to determine whether COAD and SKCM are both potential candidates for PD-L1 degradation therapies. The tumor acidic microenvironment (TAME) has been identified as a potential target for PD-L1 overexpression. Given that PD-L1 expression is positively correlated with HSPA8, a chaperone protein responsible for lysosomal degradation, it is hypothesized that targeting TAME and degrading PD-L1 through HSPA8-mediated lysosomal degradation could be an effective and precise approach to reduce PD-L1 content in whole tumor cells in SKCM and COAD.

[0005] However, traditional therapeutic drugs such as small molecule compounds, peptides, or antibodies are unable to complete this complex process because it involves the reaction of TAME outside tumor cells and the degradation of PD-L1 inside tumor cells. As a promising drug discovery approach, protein drugs can perform complex tasks, including catalyzing biochemical reactions and regulating signaling pathways. Although the rapid development of genome sequencing and structural biology has revealed an increasing number of protein blueprints with multiple functions in nature, tailoring drugs with specific protein functions remains a huge challenge today.

[0006] To achieve these research goals, researchers are working to leverage chemical engineering and synthetic biology techniques to design proteins with enhanced or expanded functionality. This involves integrating natural and / or non-natural functions into template proteins and reengineering existing natural proteins using methods such as epitope grafting, sequence randomization, and targeted screening. However, this promising strategy remains limited: not only is it hampered by the limited number of template proteins, but it also cannot fully exploit the topology of the template protein to complement the overall shape of the target protein. Therefore, a universally feasible method is needed to create natural or non-natural proteins with well-defined structures and functions as drug candidates. Summary of the Invention

[0007] The present invention aims to provide a method for synthesizing an artificial immunoglobulin and the pharmaceutical applications of the synthesized material. To explore potential candidates for tumor PD-L1 degradation therapy, the present invention analyzed a TCGA data set containing PD-L1-positive clinical samples and designed two peptide folds with specific biological functions as building blocks, each containing a left-handed and right-handed peptide pattern. Through orderly self-assembly between them, a TAME-responsive globulin-like protein nanosphere was constructed, which can bind tumor PD-L1 to HSPA8 and then deliver it to the lysosome for precise degradation. Targeting PD-L1 at TAME and degrading it through HSPA8-mediated lysosomal degradation is an effective and accurate method for reducing the PD-L1 content of entire tumor cells in SKCM and COAD. The spherical protein-like nanoartifact obtained by the present invention is named artificial immunoglobulin (IgP β) targeting PD-L1.

[0008] Based on the above objectives, the present application addresses this need in the field by providing a method for synthesizing an artificial immunoglobulin and a drug using the protein.

[0009] In one aspect, the present invention relates to a method for synthesizing an artificial immunoglobulin, wherein the artificial immunoglobulin is composed of PD-L1 LYS and PD-L1 LYS / Plus is gently self-assembled in two steps.

[0010] Furthermore, the PD-L1 of the present invention LYS and PD-L1 LYS / Plus is synthesized by solid-phase peptide synthesis method using fluoromethoxy-protected L- or D-amino acids as raw materials through HBTU / HOBT-catalyzed condensation reaction.

[0011] Specifically, the synthetic route of the present invention is as follows:

[0012] .

[0013] Specifically, the PD-L1 of the present invention LYS IgP α is self-assembled into a spherical folded body through unlimited gold-sulfur coordination; PD-L1 LYS / Plus-Au(I) precursor was added to the IgP α solution, PD-L1 LYS / Plus will self-assemble on the surface of IgP α to form the target artificial immunoglobulin IgP β under the drive of gold-philic interaction.

[0014] In another aspect, the present invention provides a drug for PD-L1 degradation, wherein the drug forms PD-L1 / PD-L1 LYS / HSPA8 complex plays a role.

[0015] Specifically, the PD-L1 of the present invention LYS It consists of three parts:

[0016] 1) A PD-L1 binding motif composed of D-enantiomer α-amino acids;

[0017] 2) triethylene glycol linker;

[0018] 3) L-enantiomer KFERQ motif.

[0019] PD-L1 LYS / Plus is an acid-responsive oligomer that binds to PD-L1 LYS An L-enantiomer HEHE motif was introduced at the carboxyl terminus of the 5′-HEXA17 protein.

[0020] The present invention also provides a drug for treating tumors, comprising an artificial immunoglobulin (IgP β) targeting PD-L1. Specifically, the drug of the present invention is particularly suitable for treating colon adenocarcinoma and skin melanoma.

[0021] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0022] 1) The present invention provides a method for synthesizing an artificial immunoglobulin and its application in medicine. The artificial immunoglobulin (IgP β) targeting PD-L1 obtained by the present invention can effectively degrade PD-L1 in the cytoplasm, cell membrane and exosomes of tumors both in vitro and in vivo, while maintaining high tumor specificity and biosafety.

[0023] 2) Drugs including those prepared by the synthetic methods of the present invention are not only effective in degrading tumor PD-L1 and achieving long-term remission of COAD and SKCM while avoiding immune-related adverse events (IrAES), but will also enable us to develop artificial protein drugs that regulate cell membrane and cytoplasmic protein content through polypeptide foldamers. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 An analysis of experimental data showing that downregulating PD-L1 may benefit patients with COAD and SKCM;

[0025] Figure 2 A roadmap for the design of artificial immunoglobulins (IgPβ) for tumor targeting.

[0026] Figure 3 Schematic diagram of the synthesis and characterization of the immunoglobulin-like nanospheres IgP β of the present invention.

[0027] Figure 4Schematic diagram of the biodistribution and biosafety analysis of IgP β of the present invention;

[0028] Figure 5 This is a schematic diagram of the analysis of IgP β lysosome-dependent degradation of tumor PD-L1 in Example 5 of the present invention;

[0029] Figure 6 This is a graph showing experimental data of Example 6 of the present invention showing that IgP β degrades tumor PD-L1 in vivo and restores T cell immunity;

[0030] Figure 7 This is a graph showing experimental data of Example 7 of the present invention showing that IgP β effectively activates cancer immunotherapy in a humanized PDOX melanoma model;

[0031] Figure 8 This is a schematic diagram showing that Example 8 of the present invention shows that IgP β can effectively inhibit tumor progression in a humanized PDOX model of colorectal cancer.

[0032] Figure 1 In the figure, AE is the bioinformatics analysis of TCGA clinical data. A is the Spearman correlation between PD-L1 expression and activated CD8+ T cells in 30 common human malignancies. B and C are the Spearman correlation scatter plots of PD-L1 expression and activated CD8+ T cells and regulatory T cells in COAD (B) and SKCM (C), respectively. D and E are the Spearman correlations between PD-L1 and immunosuppressive factor expression in COAD (D) and SKCM (E), respectively. F and G are the immunofluorescence co-staining detections of activated CD8+ T cells (CD3 + and CD8 + ) and regulatory T cells (CD4 + and CD25 + ) infiltration level. Scale bar: 50 μm. H and I show heat maps depicting changes in PD-L1, perforin-1, granzyme A, and granzyme B in the Ctrl and DR groups of PDX models of COAD (H) and SKCM (I), respectively. J shows a schematic diagram of tumor-infiltrating T cells in PD-L1-overexpressing SKCM and COAD.

[0033] Figure 2Figures A and B show the Spearman correlation between tumor PD-L1 expression and tumor-targeted markers in COAD (A) and SKCM (B), respectively. C shows the difference in HSPA8 expression between COAD tumor tissue and normal tissue. D shows the correlation between PD-L1 and HSPA8 expression in PD-L1-positive COAD. E shows the difference in HSPA8 expression between SKCM tumor tissue and normal tissue. F shows the correlation between PD-L1 and HSPA8 expression in PD-L1-positive SKCM. G shows a schematic diagram of the design and synthesis of artificial immunoglobulin IgP β.

[0034] Figure 3 In the figure, A represents the schematic diagram of the self-assembly process of IgP α and IgP β. B and C respectively represent the analysis of PD-L1 by HPLC-MS. L YS (B) and PD-L1 L YS / Plus (C). D to F indicate the TEM image (D), hydrodynamic diameter (E), and zeta potential distribution (F) of IgPα, respectively. G to I indicate the TEM image (G), hydrodynamic diameter (H), and zeta potential distribution (I) of IgPβ, respectively. J indicates an overlay of a high-resolution TEM image of IgPβ and an elemental analysis image of N, S, O, and Au from a representative particle. K and L indicate the Au 4f (K) and S 2p (L) spectra of the surface and core of IgPβ, respectively, measured by X-ray photoelectron spectroscopy (XPS). M indicates the colloidal stability of IgPβ in PBS containing 20% ​​serum at different pH values, as determined by DLS.

[0035] Figure 4 In the figure, A and B respectively indicate the expression of IgPβ in tumor-bearing C57BL / 6 mice by ICP-MS. Ctrl and accumulation of IgPβ in organs and tissues. Mice were euthanized at different time points (4 h, 10 h, 24 h, 48 h, 72 h) after systemic injection; (A) IgPβ Ctrl Dynamic distribution of IgPβ and IgPβ in organs / tissues such as heart, liver, spleen, lung, kidney, stomach, intestine and tumor. (B) IgPβ 10 hours after administration CtrlTumor-to-organ ratios of IgPβ and IgPβ. (Tu, tumor; He, heart; Li, liver; Sp, spleen; Lu, lung; Ki, kidney; St, stomach; In, intestine). C, body weight changes of C57BL / 6 mice were monitored continuously for 10 days during the experiment. D, heat map of changes in blood cells after the corresponding treatments. E, serum inflammatory cytokine concentrations (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, and TNF-α) measured by Luminex cytokine assay to reflect the immunogenicity of IgPβ in immunocompetent C57BL / 6 mice (C: control; P: IgPβ). F, liver pathological sections stained with H and E staining, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) to reflect the hepatotoxicity of IgPβ. G indicates IgPβ nephrotoxicity as assessed by H and E staining of renal pathological sections, creatinine (CRE), blood urea nitrogen (BUN), and serum albumin (ALB). H indicates IgPβ nephrotoxicity as assessed by H and E staining of lung pathological sections and changes in inflammatory factors (IFN-γ, TNF-α, IL-2, and IL-6) in lung lavage fluid. I and J indicate H and E staining of spleen (J) and heart (K), respectively. Figure 4 The data in the charts are expressed as mean ± standard deviation. Statistical analysis was performed using t-test. p <0.001. Scale bars in related figures: 50 µm.

[0036] Figure 5Figure (A) shows a schematic diagram of IgPβ targeting PD-L1 for lysosomal degradation in tumor cells. Figures (B) and (C) show immunofluorescence analysis of PD-L1 colocalization with HSPA8 (B) or LAMP (C), respectively, after 10 hours of IgPβ treatment. Fluorescence intensity distribution curves along the white line for PD-L1 and HSPA8 or LAMP, as well as the colocalization factor (Pearson's R value), were plotted using Image J. Figure (D) shows Western blot analysis of total PD-L1 expression in A375 cells after 48 hours of treatment with different concentrations of IgPβ. GAPDH was used as an internal control, and grayscale analysis was performed using ImageJ. A dose-response curve was then plotted. Figure (E) shows the effect of IgPβ on PD-L1 degradation in A375 cells. Co-staining with phalloidin (cytoskeleton) reveals that PD-L1 is degraded simultaneously at the cell membrane and in the cytoplasm (Scale bar: 100 μm; scale bar for the enlarged region: 25 μm). F indicates Western blot analysis of PD-L1 degradation in the cell membrane, cytoplasm, and exosomes in the cell culture supernatant after 48 hours of incubation with different concentrations of IgPβ. G to J indicate Western blot analysis of the effect of IgPβ on PD-L1 expression in the presence or absence of a proteasome inhibitor (MG132, 0.06μM, G), an autophagy inhibitor (3-MA, 40μM, H), and a lysosome inhibitor (NH4Cl, 250μM; chloroquine, 20μM, I and J), respectively; quantification is shown in the figure below (n=3, mean ± SD). K indicates a volcano plot of differentially expressed proteins in cells after 48 hours of IgPβ treatment. L indicates the results of gene set enrichment analysis (GSEA) of immunotherapy-related PD1 blockade, Bio Carta NFKB, and TNFR1 pathways after IgPβ treatment. *** p <0.001.

[0037] Figure 6 In the figure, A represents a schematic diagram of the B16F10 allogeneic melanoma transplant model. B represents the immunohistochemical staining of PD-L1 in mouse tumor tissue sections after corresponding treatment. C represents the expression of PD-L1 on mouse tumor exosomes, with CD63 as the internal control. D and E represent the immunofluorescence co-staining detection of CD3 in tumor tissue sections, respectively. + CD8 + (D) and CD4 + CD25 + (E) T cell infiltration (scale bar: 100 μm). F to I represent the immunohistochemical staining scores of granzyme A (F), granzyme B (G), perforin-1 (H), and CD80 (I) in the tumors of different treatment groups, respectively. J to M represent the tumor growth curves of different treatment groups, including the control group (J); IgP β, 5 mg.kg−1 (K); Ctrl IgP β (L) and Anti-PDL1, 5 mg.kg −1 (M). Data are expressed as mean ± SD, with 5 mice per group. N, Photographs of ex vivo mouse tumors after administration. O and P, tumor (O) and mouse weight (P), respectively. Q and R, representative images of H&E-stained (Q) and TUNEL-stained (R) tumor tissue sections, respectively (Scale bar: 50 μm).

[0038] Figure 7 Figure 1: A shows the construction and dosing instructions for the melanoma PDOX model. Figure 2: B shows tumor growth curves for different treatment groups (n=6 mice per group, data are presented as mean ± standard deviation). Figure 3: C shows images of tumors collected from different groups on days 1, 7, 10, 13, and at the end of the experiment. Figures 4 and 5 show tumor weight (D) and representative TUNEL staining images (E) after dosing, respectively. Figures 5 and 6 show images of PD-L1 immunohistochemistry (IHC) staining (F) and scores (G) in tumor sections, respectively. Figure 4: H shows Western blot analysis of changes in PD-L1 expression in exosomes from tumor tissue after dosing. Figures 6 and 7 show immunohistochemistry images of granzyme A (I) and perforin-1 (J), respectively. Figures 7 and 8 show the number of CD3 & CD8-positive (K) and CD4 & CD25-positive (L) T cells after treatment, respectively, as shown by immunofluorescence (Scale bar: 50 µm). p <0.05,*** p <0.001.

[0039] Figure 8 In the figure, A represents a schematic diagram of the construction of the PDOX model for colon cancer and a photo of the tumor at the end of the experiment. B to D represent the weight of the tumor after treatment (B), HE staining image (C), and TUNEL staining image (D), respectively. E represents the IHC staining image and quantitative analysis of PD-L1 in tumor tissue sections. F represents the expression of PD-L1 in tumor tissue exosomes detected by western blot after drug administration. G and H represent the immunofluorescence co-staining images of tumor sections with CD3 and CD8 (G) and CD3 and CD8 (H), respectively. I and J represent the immunohistochemical images (I) and scores (J) of granzyme A, granzyme B, perforin-1, and CD80 staining of tumor tissue, respectively. Scale bar, 50µm. p value calculated using t-test; * p <0.05;** p <0.01;*** p <0.001). DETAILED DESCRIPTION

[0040] The following is attached with the instruction manual Figure 1-8The technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0041] The experimental methods and detection methods in the following embodiments are all conventional methods unless otherwise specified; the agents and materials are all commercially available unless otherwise specified; the indicator data are all based on conventional measurement methods unless otherwise specified.

[0042] Example 1 Design of tumor-targeting artificial immunoglobulin IgP β

[0043] like Figure 2 As shown, to target overexpressed PD-L1 in tumors, this example evaluated the relationship between 18 commonly used tumor-targeting molecules and tumor PD-L1 expression in TCGA datasets (SKCM, n=472; COAD, n=459). In both SKCM and COAD, GRP68 and LAMP3, two markers of the acidic tumor microenvironment, showed the strongest correlation with PD-L1. Furthermore, the acid-responsive protein LAMP5 was found to be significantly positively correlated with PD-L1, while PD-L1 was significantly negatively correlated with GRHPR, whose expression is consistently suppressed in acidic environments. These findings suggest that the tumor acidic microenvironment is a potential target for PD-L1 overexpression. Furthermore, in COAD and SKCM, HSPA8 levels were found to be higher in tumors than in surrounding normal tissue, and PD-L1 levels were positively correlated with HSPA8 levels within the tumor site. These results suggest that targeting TAMEs in SKCM and COAD and mediated by HSPA8-mediated lysosomal degradation of PD-L1 is an effective and accurate approach to reducing PD-L1 content in entire tumor cells.

[0044] Based on this, this example uses the peptide folding body PD-L1 LYS and PD-L1 LYS / Plus constructed an immunoglobulin-like nanosphere IgPβ through a two-step mild self-assembly method. LYS and PD-L1 LYS / Plus are designed into peptide sequences by backbone engineering chiral peptide motifs. Specifically, the peptide foldomer PD-L1 that degrades PD-L1 LYS It consists of three parts: 1) a PD-L1 binding motif composed of D-enantiomer α-amino acids; 2) a tri-polyethylene glycol linker; and 3) an L-enantiomer KFERQ motif, which are involved in the delivery of chaperone-mediated lysosomal degradation. LYSWill efficiently degrade PD-L1 throughout the cell. LYS / Plus is an acid-responsive oligomer that binds to PD-L1 LYS An L-enantiomer HEHE motif was introduced at the carboxyl terminus of the 5′-HEXA17 protein.

[0045] Example 2 Structure and Characterization of Tumor-Targeting Artificial Immunoglobulin IgP β

[0046] like Figure 3 As shown, IgPβ is expressed by PDL1 LYS and PD-L1 LYS / Plus is formed through two steps of gentle self-assembly. LYS and PD-L1 LYS / Plus was first synthesized using solid phase peptide synthesis (SPPS) with fluorescein methoxy (Fmoc) protected L- or D-amino acids as raw materials via HBTU / HOBT catalytic condensation reaction. Its purity and molecular weight were then identified by liquid chromatography-mass spectrometry. LYS and PD-L1 LYS / Plus maintains product purity above 95% and their yields are all above 70%, indicating that this is a low-cost and efficient synthesis strategy. The thiol-N-terminally modified peptides in this example can form comonomer precursors through unlimited gold-sulfur coordination, and then self-assemble into spherical nanostructures driven by gold affinity. Through this self-assembly method, PD-L1 LYS IgPα can self-assemble into spherical foldamers, as evidenced by the uniform distribution of single nanoparticles imaged by transmission electron microscopy (TEM) and dynamic light scattering (DLS) characterization. In addition, the zeta potential of IgPα is 12.4 mV, which is similar to that of PD-L1. LYS Then, PD-L1 LYS / Plus-Au(I) precursor was added to the IgP α solution, PD-L1 LYS / Plus will self-assemble on the surface of IgP α driven by gold-philic interactions. Therefore, this example successfully constructed a new spherical folded body, IgP β, and the increased size of the particles and the inversion of the surface charge proved the successful construction of this structure (e.g. Figure 2 As shown in the figure). Figure 3As shown in the overlay of the elemental and TEM images, nitrogen (N), sulfur (S), oxygen (O), and gold (Au) in IgP β are uniformly distributed. In addition, X-ray photoelectron spectroscopy (XPS) of Au (4f) and S (2p) is consistent with the conjugation of Au (I) ions with thiols. This result is also confirmed by Fourier transform infrared spectroscopy (FT-IR) of IgP β.

[0047] Figure 3 The characteristic absorption peak of the gold (I)-sulfur bond is at 2950 cm -1 To test the colloidal stability of IgP β, it was diluted into PBS solutions containing 20% ​​fetal bovine serum (FBS) at three different pH values: 4.0, 6.0, and 7.4. During the 24-hour incubation, IgP β remained monodisperse with little change in hydrodynamic size, demonstrating satisfactory colloidal stability. Overall, these results demonstrate the successful construction of IgP β as protein nanospheres.

[0048] Example 3 TAME reactivity promotes tumor accumulation of IgPβ

[0049] Figure 4 As shown, PD-L1 LYS The HEHE motif in the / Plus peptide confers additional charge reversal capability to IgPβ in response to TAME. To investigate the significance of TAME reactivity in IgPβ, this example used the same synthetic pathway as IgPβ to synthesize PD-L1. LYS Replaces PDL1 LYS / Plus constructed a TAME-insensitive nanosphere (IgPβ Ctrl As expected, IgPβ showed electronegativity at pH >7.0 and electropositivity at pH <7.0, whereas IgPβ Ctrl It maintains positive charge in both alkaline and acidic environments. Therefore, this acid responsiveness can make IgP β selective for cellular internalization in response to pH in the extracellular microenvironment. As expected, at pH 6.5, which simulates the acidic microenvironment of tumors, IgP β has a strong cellular internalization ability in three tumor cell lines (B16F10, HCT116 and NCI-H1975), while the cellular internalization effect is weakened at pH 7.4. In addition, acid responsiveness reduces drug accumulation in the reticuloendothelial system (liver and spleen) and digestive system (stomach and intestines) after intravenous injection, indicating an optimized pharmacokinetic profile of IgP β. More importantly, compared with IgP β CtrlIn contrast, IgPβ tumor accumulation increased 3.1-fold, which is attributed to enhanced tumor targeting by TAME reactivity, further demonstrating that IgPβ has a more pronounced tumor accumulation tendency than IgPβCtrl. In summary, TAME reactivity promotes tumor accumulation of IgPβ, a favorable feature for improving drug efficacy and reducing toxicity.

[0050] Example 4 IgP β has good safety

[0051] Figure 4 As shown, to test the biosafety of IgPβ, 12 healthy C57BL / 6 mice were intravenously injected with 200 μL normal saline (NS) every 2 days as a control group or an equal volume of 10 mg / kg IgPβ as an experimental group (n = 6 / group). After 10 days of administration, there was no significant difference in body weight between the two groups of mice. In addition, routine blood analysis after administration showed no obvious adverse reactions such as hemolysis, bone marrow suppression, anemia, leukopenia, and thrombocytopenia. In order to explore the allergic reaction of IgPβ, we measured a series of serum biochemical indicators involved in immunotoxicity, including IFNγ, IL1β, IL2, IL4, IL5, IL6, IL10, IL12 and TNFα, 10 days after administration, and no immunotoxicity was observed. In addition, Figure 4 Pathological examinations of healthy livers, aspartate aminotransferase (ALT), alanine aminotransferase (AST), and total bilirubin (TBiL) in the liver, and of kidneys, blood urea nitrogen (BUN), creatinine (CREA), and albumin (ALB) in the kidneys, demonstrated that IgPβ was non-hepatotoxic and non-nephrotoxic. Furthermore, compared with the NS group, IgPβ administration had little effect on lung morphology and levels of IFNγ, TNFα, IL-2, and IL-6 in lung lavage fluid. Furthermore, no pathological morphology was observed in HE sections of the spleen and heart. Taken together, these data demonstrate that IgPβ is sufficiently non-toxic for clinical translation.

[0052] Example 5 IgPβ effectively degrades tumor PD-L1 through lysosomal degradation involving HSPA8

[0053] like Figure 5 As shown. According to the design, IgP β can be broken down into PD-L1 under the action of high concentrations of glutathione in cells. LYS and PD-L1 LYS / plus monomers, then, PD-L1 in the cytoplasm LYS As a catalyst, it downregulates PD-L1 through HSPA8-derived lysosomal degradation. Molecular docking results showed that PD-L1 LYSIt can bind to both PD-L1 and HSPA8 simultaneously. Colocalization analysis of PD-L1 and HSPA8 in A375 cells confirmed this result. After 10 hours of incubation with IgP β, significant colocalization was observed between red-labeled PD-L1 and green-labeled HSPA8, while almost no colocalization was observed in the no-drug control. In theory, HSPA8 can bind to both PD-L1 and PD-L1. LYS The PD-L1 / HSPA8 complex is recruited to the lysosome for degradation. To verify this, we performed colocalization analysis of PD-L1 and Lamp1 (a lysosomal marker) in A375 cells. Increased colocalization was observed after IgPβ treatment, suggesting that IgPβ can induce PD-L1 to enter the lysosome. Furthermore, colocalization of PD-L1 / HSPA8 and PDL1 / Lamp1 was reaffirmed in IgPβ-treated HCT116 cells. Consequently, IgPβ induced the degradation of total PD-L1 in a dose-dependent manner, with a median inhibitory concentration (IC50) of 0.86 ± 0.16 μM. Furthermore, immunofluorescence staining and western blot results showed that IgPβ downregulated not only cytoplasmic PD-L1 but also membrane PD-L1. Similar results were observed in HCT116 cells. More importantly, Western blot and protein spectrometry (LC-MS) revealed that IgPβ statistically significantly reduced the amount of PD-L1 in exosomes. To further validate the mechanism of PD-L1 degradation, this example used the proteasome inhibitor MG132, the autophagosome inhibitor 3-MA, and the lysosomal inhibitors NH4Cl and chloroquine to inhibit PD-L1 degradation. As expected, MG132 and 3-MA had little effect on IgPβ-induced PD-L1 degradation, while NH4Cl and chloroquine inhibited the IgPβ-induced PD-L1 degradation. These results suggest that IgPβ induces PD-L1 degradation through a lysosome-dependent protein degradation pathway. Furthermore, after 10 hours of incubation in B16F10 cells, IgPβ triggered the proteomic expression of 1006 differentially expressed proteins. Further gene set enrichment analysis (GSEA) revealed that, compared with mock-treated cells, IgPβ-treated cells showed significant upregulation of gene sets related to cancer immunotherapy, including PD1 blockade, the NFKB pathway, and the TNFα pathway, further suggesting a role for IgPβ in PD-L1 degradation. In summary, IgPβ effectively degrades PD-L1 in the cytoplasm, cell membranes, and exosomes in a lysosome-dependent manner.

[0054] Example 6 IgPβ degrades tumor PD-L1 in vivo and restores T cell immunity

[0055] like Figure 6As shown in Figure 2, in order to study the role of IgPβ in degrading PD-L1 in vivo, this example established a C57BL / 6 mouse subcutaneous implantation of a syngeneic B16F10 melanoma model. When the tumor volume reached 100±20 mm 3 20 mice were randomly divided into 4 groups: (1) blank control group NS Control, (2) experimental group IgP β, (3) system control group, PEG was used instead of peptide, Ctrl IgPβ, (4) positive control group, mouse Anti-PD-L1. It is worth noting that one treatment cycle includes one day of intravenous injection of the drug with an injection volume of 100μL and a dose of 5mg / Kg and four days of treatment observation. After two treatment cycles, IgPβ significantly reduced the content of PD-L1 in tumor tissues, while Ctrl IgP β and Anti-PD-L1 did not show similar effects. Ctrl In stark contrast to IgPβ or Anti-PD-L1, IgPβ also caused a significant downregulation of PD-L1 in exosomes. These results indicate that IgPβ can effectively degrade PD-L1 in the cytoplasm, cell membrane, and exosomes in vivo. Compared with Anti-PD-L1, IgPβ is more effective in promoting CD8 + Cytotoxic T lymphocyte (CTL) infiltration and reduced the number of regulatory T cells in the tumor. Ctrl Compared with IgP β, IgP β significantly increased the activity of intratumoral CTLs, which was manifested by a significant upregulation of granzyme A, granzyme B, perforin-1 and CD80 in the tumor site. Therefore, after two simultaneous treatment cycles, the ability of IgP β to inhibit tumor growth was 92.00%, and its anti-tumor effect was stronger than the 57.15% of the Anti-PD-L1 group. The tumor photos and direct measurement of tumor weight at the end of the experiment were consistent with the conclusions of the measured tumor volume. It is worth noting that compared with the control group treated with mock treatment, the tumors treated with IgP β or Ctrl There was no statistically significant difference in body weight between IgPβ-treated mice, demonstrating the inherent biosafety of the immunoglobulin-like nanospheres. Furthermore, a significant increase in apoptosis in tumor cells treated with IgPβ was observed in staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) assays. In summary, IgPβ effectively degrades tumor PD-L1 in vivo and activates anti-tumor T cell immunity.

[0056] Example 7 IgPβ can effectively activate cancer immunotherapy in the SKCM humanized PDOX model

[0057] like Figure 7As shown, orthotopic xenograft (PDOX) tumors derived from patients with humanized immune systems are closely related to the heterogeneity and immune microenvironment of human cancers and are the first choice for preclinical research of new immunotherapy drugs. To study the therapeutic effect of IgP β, this example excised malignant melanoma tissue with high PD-L1 expression obtained by surgery and transplanted into the right axillary skin mesenchyme of 18 female completely immunodeficient NOD / SCID mice. To reconstitute immunity, humanized peripheral blood mononuclear cells (PBMCs) donated by a 30-year-old healthy woman were injected at a rate of 1×10 6 The mice were intravenously injected with a dose of 100 μL / mouse. When the PDX tumor grew to 50±10 mm3 (day 15 after tumor transplantation), 18 mice were randomly divided into a control group (NS, 100 μL), an experimental group (IgP β, 5 mg / Kg), and a positive control group (Anti-PD-L1, 5 mg / Kg). Figure 7 The treatment regimen shown was started two weeks after tumor transplantation and continued for two weeks. After administration, the PDOX tumors in the control group had reshaped humanized immunity, but the tumor volume had grown to approximately 1800 mm 3 , suggesting that the tumor possesses immune resistance. In this example, IgPβ treatment was highly effective, with a tumor growth inhibition rate (TGI) of 80.55%, significantly higher than the 51.03% inhibition rate achieved with anti-PD-L1. Ex vivo tumor images and TUNEL staining further confirmed the high efficacy of IgPβ. Consistent with these results, IgPβ effectively degraded PD-L1 in tumor tissue and tumor exosomes. Furthermore, IgPβ upregulated the levels of CTL-associated proteins such as granzyme A, granzyme B, perforin-1, and CD80 at the tumor site, increasing intratumoral CTL infiltration and reducing the number of immunosuppressive regulatory T cells. Taken together, these results demonstrate that IgPβ can effectively activate immunotherapy for PD-L1-positive malignant melanoma and inhibit tumor progression.

[0058] Example 8 IgPβ can effectively inhibit tumor progression in the COAD humanized PDOX model

[0059] like Figure 8 To further investigate the role of IgPβ, we orthotopically implanted tumors derived from surgically resected colorectal cancer patients into the submesenteric tissue of the colon of NOD / SCID mice to establish a PDOX model of colorectal cancer. 6Nine mice successfully modeled with human PBSCs were intravenously injected at a dose of 100 cells / mouse to reconstitute humanized immunity. The mice were randomly divided into a control group (NS, 100 μL), an experimental group (IgP β, 5 mg / kg), and a positive control group (anti-PD-L1, 5 mg / kg). Each group received two intravenous injections of the drugs over a two-week period. Tumor images, tumor weights, pathological H&E sections, and TUNEL staining revealed that anti-PD-L1 exhibited very limited efficacy against these PDOX tumors, whereas IgP β exhibited robust efficacy against colorectal cancer after tumor immune escape. Furthermore, IgP β, as expected, degraded PD-L1 in tumor tissue and tumor exosomes, further confirming the robust and potent biological function of IgP β. Furthermore, the absence of infiltrating CTLs, the accumulation of immunosuppressive Treg cells, and the loss of CTL-associated proteins granzyme A, granzyme B, perforin-1, and CD80 in tumors all indicate features of tumor immune escape. The results of this example show that IgPβ successfully activated T cell immunity in colorectal cancer, while PD-L1 neutralizing monoclonal antibodies showed very limited immunotherapy ability. In summary, IgPβ can activate the tumor immunity function of colorectal cancer that escapes innate immunity and inhibit tumor development.

[0060] This example introduces chiral peptide motifs into the peptide sequence through the backbone based on the basic construction principle of peptide foldamers and peptide backbone engineering, and designs two synthetic oligomers. LYS and PD-L1 LYS Under the influence of the gold-affinity interaction of the Au-peptide precursor, IgP / Plus self-assembles into immunoglobulin-like protein nanospheres with anti-tumor immune activation. IgP / Plus exhibits excellent pH responsiveness, cell membrane permeability, preferential tumor aggregation, and a favorable biosafety profile (no immunogenicity or acute toxicity). As expected, IgP / Plus effectively degrades PD-L1 in the cytoplasm, cell membranes, and exosomes in a lysosome-dependent manner. Importantly, compared with PD-L1 antibodies, IgP / Plus exhibits superior therapeutic efficacy in a C57BL / 6 mouse syngeneic model of melanoma and in a humanized mouse PDOX model of PD-L1-overexpressing malignant melanoma and immune-resistant colorectal cancer. This invention expands the application of peptide foldamers to the discovery of artificial protein drugs for lysosomal-targeted degradation of membrane and cytoplasmic proteins and has the potential to revitalize ICB therapy, making it a more widespread and effective treatment option to improve the prognosis of patients with advanced cancer.

[0061] As described above, the present invention can be better implemented. The above embodiments only describe the preferred implementation methods of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A method for synthesizing an artificial immunoglobulin, characterized in that: The artificial immunoglobulin is composed of PD-L1 LYS and PD-L1 LYS / Plus is made through two-step gentle self-assembly; ； PD-L1 LYS IgP α is self-assembled into a spherical folded body through unlimited gold-sulfur coordination; PD-L1 LYS / Plus-Au(I) precursor was added to the IgP α solution, PD-L1 LYS / Plus will self-assemble on the surface of IgP α to form the target artificial immunoglobulin IgP β under the drive of gold-philic interaction.

2. The method for synthesizing artificial immunoglobulin according to claim 1, wherein PD-L1 LYS and PD-L1 LYS / Plus is synthesized by solid-phase peptide synthesis method using fluoromethoxy-protected L- or D-amino acids as raw materials through HBTU / HOBT-catalyzed condensation reaction.

3. A drug for PD-L1 degradation, characterized in that: The drug is PD-L1 LYS / Plus self-assembles on the surface of IgP α under the drive of gold-philic interaction to form the artificial immunoglobulin IgP β according to claim 1.

4. The PD-L1 degradation drug according to claim 3, characterized in that PD-L1 LYS It consists of three parts: 1) A PD-L1 binding motif composed of D-enantiomer α-amino acids; 2) triethylene glycol linker; 3) L-enantiomer KFERQ motif.

5. A drug for treating colon adenocarcinoma, characterized in that: The medicine contains a substance synthesized by the method according to any one of claims 1-2.

6. A drug for treating skin melanoma, characterized in that: The medicine contains a substance synthesized by the method according to any one of claims 1-2.

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