Tumor-specific melittin / antibody double-blocking prodrug

Abstract

The invention provides a tumor-specific melittin / antibody double-blocking prodrug molecule and application thereof, the prodrug molecule is in a peripheral tissue environment, the activity of an antibody and melittin in the prodrug molecule is masked, and a double-blocking state is formed; in a tumor microenvironment, specific enzyme in the tumor microenvironment can cut the cleavable peptide chain in the connecting fragment, so that the antibody and the melittin in the prodrug molecule are released and the activity of the antibody and the melittin is recovered, and the antibody and the melittin are in a double-activation state. The double-blocking prodrug molecule provided by the invention effectively solves the problem of non-targeting toxicity of the antibody, can exert different anti-tumor mechanisms of toxin and the antibody, achieves a synergistic treatment effect, and improves the curative effect and the safety at the same time.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese application No. 202510515994.9 filed on April 23, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention belongs to the field of biomedicine, and in particular relates to a tumor-specific melittin / antibody dual-blocking prodrug and its application. Background Art

[0004] A tumor is a new growth formed when cells in local tissues lose normal genetic regulation of their growth under the influence of various tumorigenic factors, leading to clonal abnormal proliferation. Once formed, a new growth does not stop even if the cause is eliminated. Its growth is not subject to normal physiological regulation and instead destroys normal tissues and organs.

[0005] Tumor cells have multiple mechanisms to evade recognition and attack by the immune system, which is a key foundation for tumor development. Immune-stimulating drugs primarily activate the body's own immune system in various ways to attack tumor cells, disrupting this immune evasion mechanism.

[0006] Although immune-agonistic drugs such as immune cell receptor complex antibodies (such as CD3 antibodies), immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies), and co-stimulatory molecule agonists (such as 4-1BB antibodies) have made revolutionary progress in cancer treatment, they still face challenges such as insufficient efficacy and immune-related adverse events (irAEs), which severely limit their application in patients, especially those with autoimmune diseases.

[0007] Peptide toxins are a class of cytotoxic molecules composed of polypeptides. Their sources include animal (e.g., snake venom, melittin), plant (e.g., ricin), and bacterial (e.g., diphtheria toxin, tetanus toxin). These toxins kill tumor cells through various mechanisms, including direct interference with cellular physiological processes, induction of apoptosis, or enhanced therapeutic efficacy through immune responses. Melittin, a 26-amino acid cationic peptide, exhibits significant antitumor activity. Its antitumor mechanisms involve multiple actions, including direct tumor cell killing, modulation of the immune microenvironment, and inhibition of tumor metastasis. Melittin's amphipathic structure (a hydrophobic end with a positively charged hydrophilic end) can insert into the phospholipid bilayer of tumor cell membranes, forming transmembrane pores that lead to ion leakage and cell lysis. Melittin can also induce macrophage phenotype transformation from M2 (pro-tumor) to M1 (anti-tumor) phenotype, enhancing phagocytosis. Furthermore, by releasing tumor antigens (e.g., HMGB1), it promotes dendritic cell (DC) maturation and T cell activation. Studies have shown that the combination of bee venom and PD-1 / PD-L1 antibodies can further enhance the anti-tumor effect.

[0008] However, melittin lacks tumor targeting. While killing tumor cells, it may also have adverse effects on normal tissues and cells, such as hemolysis and liver and kidney damage. Reducing the toxicity of melittin to normal tissues and improving its tumor targeting are key issues that need to be addressed in clinical applications.

[0009] Therefore, there is an urgent need to develop new strategies to address the tumor targeting of immune-agonistic antibodies and toxin molecules to improve safety, while also enhancing tumor treatment efficacy through combined use. Summary of the Invention

[0010] The present invention provides a tumor-specific melittin / antibody dual-blocking prodrug molecule and application thereof.

[0011] In a first aspect of the present invention, a dual-blocking prodrug molecule is provided, comprising: (a) a first active component, (b) a second active component, and (c) a coupling element located between the first active component and the second active component for coupling the first active component and the second active component together; wherein the first active component is a toxin molecule, and the second active component is a functional polypeptide or protein having a predetermined activity;

[0012] Furthermore, the prodrug molecule has a first state. When the prodrug molecule is in the first state (or blocking state), the first active component is in a toxicity blocking state, and the activity of the second active component is blocked by the first active component and / or the coupling element.

[0013] Furthermore, when the prodrug molecule is cleaved, it is converted into a drug having a second state; in the second state, the first active component and the second active component are separated, and the first active component regains its toxicity while the second active component regains its activity.

[0014] In another preferred embodiment, the coupling element of the prodrug molecule is cut, so that after the first active component and the second active component are separated, the first active component recovers its toxicity, and the activity of the second active component is no longer blocked by the first active component and / or the coupling element, thereby recovering its activity.

[0015] In another preferred embodiment, the dual-blocking prodrug molecule is cleaved by an enzyme in the body of the administered subject, thereby releasing the first active component and the second active component having respective activities.

[0016] In another preferred embodiment, the dual-blocking prodrug molecule is cleaved at or around the lesion site.

[0017] In another preferred embodiment, in the first state (or activity-blocked state), the activity of the first active component is blocked by the second active component and / or the coupling element.

[0018] In another preferred embodiment, the functional polypeptide or protein having a predetermined activity comprises a functional polypeptide or protein having one or more predetermined activities.

[0019] In another preferred embodiment, the one or more include 1, 2, 3, or 4 types.

[0020] In another preferred embodiment, the prodrug molecule has a first state and a second state, the first state is an activity blocking state, and the activity blocking state means that the activity of the active component in the prodrug molecule is masked in the non-lesion site; the second state is an activity activation state, and the activity activation state means that the active component in the prodrug molecule is released and restores activity in the lesion site.

[0021] In another preferred embodiment, the lesion site is a tumor microenvironment.

[0022] In another preferred embodiment, the active component comprises a first active component and a second active component.

[0023] In another preferred embodiment, in a non-tumor microenvironment, the activities of the first active component and the second active component in the prodrug molecule are masked, forming a dual-blocking state;

[0024] In the tumor microenvironment, specific enzymes in the tumor microenvironment cleave the cleavable peptide chain in the connecting fragment, and the first active component and the second active component are released and restored to activity, thereby being in a dual-activated state.

[0025] In another preferred embodiment, the non-tumor microenvironment includes: peripheral tissue environment.

[0026] In another preferred embodiment, the first active ingredient is a toxin.

[0027] In another preferred embodiment, the second active component is a functional polypeptide or protein.

[0028] In another preferred embodiment, the activity being masked includes: the activity being significantly inhibited or completely inhibited.

[0029] In another preferred embodiment, the prodrug molecule comprises the structure shown in Formula I:

[0030] Z1-Z2-Z3(I)

[0031] wherein Z1 is the first active component, and the first active component is a toxin;

[0032] Z2 is a coupling element comprising an enzyme-specific cleavable peptide chain and / or a polypeptide sequence containing a hinge region; and

[0033] Z3 is the second active component, which is a functional polypeptide or protein.

[0034] In another preferred embodiment, the functional polypeptide or protein is a functional polypeptide or protein having a predetermined activity.

[0035] In another preferred embodiment, the toxin is a polypeptide toxin or a mutant thereof.

[0036] In another preferred embodiment, the toxins include: animal-derived toxins, plant-derived toxins, bacterial-derived toxins, and / or toxins from other sources.

[0037] In another preferred embodiment, the toxin comprises: toad venom, snake venom, scorpion venom, centipede venom, spider venom, ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, triptolide, trichosanthin, bee venom, or a combination thereof.

[0038] In another preferred embodiment, the toxin is melittin.

[0039] In another preferred embodiment, the functional polypeptide or protein is a tumor cell killer or an immune agonist. In another preferred embodiment, the functional polypeptide or protein comprises: an antibody or a toxin.

[0040] In another preferred embodiment, the prodrug molecule comprises the structure shown in Formula I:

[0041] Z1-Z2-Z3(I)

[0042] Among them, Z1 is toxin;

[0043] Z2 is a coupling element comprising an enzyme-specific cleavable peptide chain and / or a polypeptide sequence containing a hinge region; and

[0044] Z3 is an antibody.

[0045] In another preferred embodiment, the antibody is a tumor-specific antibody.

[0046] In another preferred embodiment, the antibody is a tumor-specific immune agonist.

[0047] In another preferred embodiment, the antibody comprises: a monospecific antibody, a bispecific antibody, or a trispecific antibody.

[0048] In another preferred embodiment, the antibodies include: monoclonal antibodies, polyclonal antibodies, double-chain antibodies, single-chain antibodies (scFv), Fab, Fab', and F(ab')2 antibodies.

[0049] In another preferred embodiment, the antigen of the antibody comprises: an immune checkpoint, a co-stimulatory molecule, an immune receptor complex, a cytokine, or a tumor antigen.

[0050] In another preferred embodiment, the antibodies include: CD3 antibody, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, 4-1BB antibody, CD28 antibody, CD47 antibody, OX40 antibody, ICOS antibody, CD27 antibody, CXCL12 antibody, Her2 antibody, EGFR antibody, or VEGF antibody.

[0051] In another preferred embodiment, the functional polypeptide or protein is a fusion protein.

[0052] In another preferred embodiment, the fusion protein comprises two, three, or four functional elements.

[0053] In another preferred embodiment, the functional element comprises: integrin binding peptide, collagen binding peptide, cytokine, or chemokine.

[0054] In another preferred embodiment, the cytokine comprises interleukin molecules IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

[0055] In another preferred embodiment, the functional element improves the tissue distribution of the prodrug molecule and enhances its targeting to tumor or inflammatory tissue.

[0056] In another preferred embodiment, the coupling element comprises a structure shown in Formula II:

[0057] Z21-Z22-Z23(II)

[0058] Wherein, the Z21 is an enzyme-specific cleavable peptide chain;

[0059] Z22 is a polypeptide sequence containing a hinge region; and

[0060] The Z23 is an enzyme-specific cleavable peptide chain.

[0061] In another preferred embodiment, the enzyme-specific cleavable peptide chain comprises a tumor-specific enzyme-cleavable peptide chain.

[0062] In another preferred embodiment, the enzyme-specific cleavable peptide chain comprises: a uPA enzyme-specific cleavable peptide chain, a MMP enzyme-specific cleavable peptide chain, an AEP enzyme-specific cleavable peptide chain, or a combination thereof.

[0063] In another preferred embodiment, the enzyme-specific cleavable peptide chain is a uPA enzyme-specific cleavable peptide chain.

[0064] In another preferred embodiment, the enzyme-specific cleavable peptide chain is a MMP enzyme-specific cleavable peptide chain.

[0065] In another preferred embodiment, the Z21 and the Z23 are peptide chains cleavable by different enzyme specificities.

[0066] In another preferred embodiment, the Z21 and the Z23 are the same enzyme-specific cleavable peptide chain.

[0067] In another preferred embodiment, the hinge region-containing polypeptide sequence comprises: an IgG hinge region, an IgA hinge region, an IgM hinge region, or a combination thereof.

[0068] In another preferred embodiment, the hinge region-containing polypeptide sequence is a human IgG1 hinge region.

[0069] In another preferred embodiment, the prodrug molecule comprises the prodrug molecule MuHuA: melittin-uPA enzyme-specific cleavable peptide chain-IgG hinge region-uPA enzyme-specific cleavable peptide chain-atezolizumab.

[0070] In another preferred embodiment, the prodrug molecule comprises the prodrug molecule MmHm4R: melittin MP5 mutant-MMP enzyme-specific cleavable peptide chain-IgG hinge region-MMP enzyme-specific cleavable peptide chain-4-1BB antibody.

[0071] In another preferred embodiment, when the enzyme corresponding to the enzyme-specific cleavable peptide chain is not present, the activities of Z1 and Z3 are masked;

[0072] When the enzyme corresponding to the enzyme-specific cleavable peptide chain is present, Z1 and / or Z3 are released and their activity is restored.

[0073] In another preferred embodiment, when the enzyme corresponding to the enzyme-specific cleavable peptide chain is not present, the toxicity of the toxin is masked, and the activity of the functional polypeptide or protein is masked;

[0074] When the enzyme corresponding to the enzyme-specific cleavable peptide chain is present, the toxin is released and its toxicity is restored, and / or the functional polypeptide or protein is released and its activity is restored.

[0075] In another preferred embodiment, the prodrug molecule masks or significantly inhibits the activities of the antibody and melittin in the peripheral tissue environment, forming a double-blocking state;

[0076] In the tumor microenvironment, the uPA enzyme in the tumor microenvironment will cut the cleavable peptide chain in the connecting fragment, and both the antibody and bee venom are released and restored to activity, thus being in a dual-activated state.

[0077] In another preferred embodiment, the activity of the prodrug molecule in the blocked state is significantly increased compared to the activity of the prodrug molecule in the activated state.

[0078] In another preferred embodiment, the activity includes: hemolytic activity, immune-related adverse reactions, and / or tumor cell-killing activity.

[0079] In another preferred embodiment, the hemolytic activity is characterized by an EC50 value.

[0080] In another preferred embodiment, the significant increase means that the ratio of the EC50 value A1 of the prodrug molecule in the active masked state to the EC50 value A2 of the prodrug molecule after the active toxin and / or antibody is released and the activity is restored is A1 / A2≥50, preferably A1 / A2≥500.

[0081] In another preferred embodiment, the tumor cell killing activity is characterized by the IC50 value of lysing tumor cells.

[0082] In another preferred embodiment, the significant increase means that the ratio of the IC50 value B1 of the prodrug molecule in the active masked state to the IC50 value B2 of the prodrug molecule after the active toxin and / or antibody is released and the activity is restored is B1 / B2≥10, preferably B1 / B2≥50.

[0083] In another preferred embodiment, the immune-related adverse reactions include: suppression or killing of immune cells, non-specific activation of immune cells, fever, joint pain, interstitial pneumonia, hepatitis, enteritis, edema, or a combination thereof.

[0084] In another preferred embodiment, the immune cells are suppressed or killed, including: a decrease in red blood cell count, a decrease in granulocyte count, a decrease in platelet count, or a combination thereof.

[0085] In another preferred embodiment, the non-specific activation of the immune cells includes: an increase in T cells, an increase in NK cells, an increase in granulocytes, or a combination thereof.

[0086] In a second aspect of the present invention, a recombinant protein or polypeptide is provided, comprising:

[0087] (i) the prodrug molecule according to the first aspect of the present invention;

[0088] (ii) Optional functional polypeptide or protein.

[0089] In another preferred embodiment, the functional polypeptide or protein improves the targeting to tumor or inflammatory tissue.

[0090] In another preferred embodiment, the functional polypeptide or protein comprises: an integrin binding peptide, a collagen binding peptide, a cytokine, or a chemokine.

[0091] In another preferred embodiment, the cytokine comprises interleukin molecules IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

[0092] In another preferred embodiment, the functional polypeptide or protein is RGD polypeptide.

[0093] In the third aspect of the present invention, a polynucleotide is provided, which encodes the prodrug molecule described in the first aspect of the present invention, or the recombinant protein or polypeptide described in the second aspect of the present invention.

[0094] In the fourth aspect of the present invention, a vector is provided, wherein the vector comprises the polynucleotide according to the third aspect of the present invention.

[0095] In another preferred embodiment, the vector comprises a eukaryotic cell expression vector or a prokaryotic cell expression vector.

[0096] In another preferred embodiment, the vector is a plasmid.

[0097] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell comprises the vector according to the fourth aspect of the present invention or the polynucleotide according to the third aspect of the present invention is integrated into its genome.

[0098] In another preferred embodiment, the host cell comprises a eukaryotic cell or a prokaryotic cell.

[0099] In the sixth aspect of the present invention, a pharmaceutical composition is provided, comprising the prodrug molecule according to the first aspect of the present invention.

[0100] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0101] In the seventh aspect of the present invention, there is provided a use of the prodrug molecule described in the first aspect of the present invention, the recombinant protein or polypeptide described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the host cell described in the fifth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention for preparing a drug kit for treating tumors.

[0102] In another preferred embodiment, the tumor treatment includes: highly selectively killing tumors.

[0103] In another preferred embodiment, the highly selective tumor killing means that the killing effect on tumors is at least 10 times that on healthy tissues, preferably at least 50 times, and most preferably at least 100 times.

[0104] In the eighth aspect of the present invention, a medicine kit is provided, which comprises the prodrug molecule described in the first aspect of the present invention, the recombinant protein or polypeptide described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the host cell described in the fifth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention.

[0105] In another preferred embodiment, the medicine kit further comprises an instruction manual, wherein the instruction manual is used to guide the use of the medicine kit for treating tumors.

[0106] In another preferred embodiment, the drug kit further comprises a diagnostic reagent for tumors.

[0107] In another preferred embodiment, the drug kit further comprises: other optional tumor therapeutic agents.

[0108] In a ninth aspect of the present invention, there is provided a method for activating the prodrug molecule according to the first aspect of the present invention, comprising the following steps:

[0109] (S1) providing the prodrug molecule described in the first aspect of the present invention;

[0110] (S2) activating the prodrug molecule of the first aspect of the present invention in the presence of an enzyme corresponding to the enzyme-specific cleavable peptide chain, thereby producing a toxin and / or an antibody.

[0111] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.

[0112] In a tenth aspect of the present invention, a reaction system for in vitro activation of the prodrug molecule described in the first aspect of the present invention is provided, wherein the reaction system comprises:

[0113] (i) the prodrug molecule according to the first aspect of the present invention; and

[0114] (ii) a sample comprising an enzyme corresponding to the enzyme-specific cleavable peptide chain in the prodrug molecule according to the first aspect of the present invention.

[0115] In another preferred embodiment, the sample includes: peripheral blood sample, pleural effusion, ascites, or cerebrospinal fluid.

[0116] In another preferred embodiment, the sample is an enzyme solution prepared in a laboratory.

[0117] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 It was shown that MuHuA blocks melittin activity in vitro and releases active melittin after cleavage.

[0119] Specifically, Figure 1 A shows a schematic diagram of the design of the MuHuA prodrug, in which bee venom peptide and the hinge region of human IgG1 antibody are fused to the N-termini of the heavy and light chains of atezolizumab through two uPA-cleavable linker peptides. Figure 1 B shows reducing SDS-PAGE analysis of atezolizumab, MuHuA or MHA, and uPA-cleaved MuHuA or MHA (A: atezolizumab; HC: heavy chain; LC: light chain). Figure 1 C shows the hemolytic activity of melittin (mel), melittin whose activity is masked in MuHuA (Mel locked), and melittin released from MuHuA (Mel released) on mouse erythrocytes.

[0120] Figure 2 The lytic activities of chemically synthesized prototype melittin, melittin whose activity was masked in MuHuA, and melittin released from MuHuA on MC38 tumor cells are shown.

[0121] Figure 3 MuHuA blocks the hemolytic activity of melittin in vitro. Mouse red blood cells (RBCs) were incubated with graded concentrations of chemically synthesized melittin, melittin whose activity was masked in MuHuA, or melittin released from MuHuA for 30 minutes at 37°C. RBCs treated with 1% Triton X-100 and PBS served as positive (Pos) and negative (Neg) controls, respectively.

[0122] Figure 4The results show that MuHuA releases active melittin after in vitro cleavage. The lytic activity of free melittin (mel), melittin locked in MuHuA, and melittin released from MuHuA on HT29 cells.

[0123] Figure 5 It was shown that MuHuA reduces the toxic side effects of melittin in vivo.

[0124] Specifically, Figure 5 A shows the sublethal toxicity experimental scheme. Figure 5 B shows the daily changes in body weight of mice after injection. Figure 5 C shows blood samples collected 4 days after injection, and the number of red blood cells in the blood was measured by routine blood tests. Figure 5 D shows blood samples collected 4 days after injection, and plasma alanine aminotransferase (ALT) (left) and aspartate aminotransferase (AST) levels (right) were measured by chemical analysis. Figure 5 E shows the lethal toxicity experimental scheme. Figure 5 F shows the survival of mice monitored after injection. All graphs are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[0125] Figure 6 MuHuA caused less hemolysis than free melittin. Blood samples were collected from mice in each group on day 4 after drug injection. Hemoglobin (HGB) concentration and platelet (PLT) count in the blood were measured by routine blood tests.

[0126] Figure 7 ELISA detection of binding of atezolizumab (Ate), activity-masked Ate in HuA, activity-masked Ate in MuHuA, and Ate released from MuHuA to plate-immobilized mouse PD-L1 is shown.

[0127] Figure 8 The blood glucose levels of mice were monitored daily after injection. Blood glucose levels ≥ 13.88 mmol / L were considered diabetic.

[0128] Figure 9 Shown are pancreatic samples collected 11 days after injection, and the expression levels of several inflammation-related cytokines in the pancreas were measured using quantitative real-time PCR.

[0129] Figure 10 The plasma concentrations of human IgG (hIgG), atezolizumab (Ate), and MuHuA during the distribution phase (left) and elimination phase (right) as measured by ELISA are shown, indicating that MuHuA has better pharmacokinetic properties than Atz.

[0130] Figure 11 The plasma concentrations of Ate, MuHuA, and Ate released from MuHuA in normal organs (left) and tumors (right) detected by ELISA are shown, indicating that MuHuA has better tissue grouping characteristics than Ate.

[0131] Figure 12 MuHuA was shown to exhibit potent antitumor effects in a thermal tumor model without causing toxicity.

[0132] Specifically, Figure 12 A shows the experimental scheme of the MC38 tumor model. Figure 12 B shows the tumor growth curve. Figure 12 C shows the survival rate.

[0133] Figure 13 The results show that on the 22nd day after tumor inoculation, the tumor tissues of mice in each group treated with different drugs were collected to prepare single cell suspensions, and the tumor infiltrating lymphocytes (CD45 + , TIL) T cells (CD45 + CD3 + ) ratio.

[0134] Figure 14 The results show that on the 22nd day after tumor inoculation, the tumor tissues of mice in each group treated with different drugs were taken to prepare single cell suspensions, and T cells (CD45 + CD3 + ) + T cells (CD45 + CD3 + CD8 + ) ratio.

[0135] Figure 15 The results show that on the 22nd day after tumor inoculation, the tumor tissues of mice in each group treated with different drugs were taken to prepare single cell suspensions, and non-T cells (CD45 + CD3 - ) in NK cells (CD45 + CD3 - CD335 + ) ratio.

[0136] Figure 16 Blood samples were collected 22 days after tumor inoculation, and the number of red blood cells in the blood was measured by routine blood tests.

[0137] Figure 17MuHuA significantly reduces the hemolytic side effects of melittin. Blood samples were collected on day 22 after tumor inoculation. Hemoglobin (HGB) concentration (left) and platelet (PLT) count (right) in the blood were measured by routine blood tests.

[0138] Figure 18 It was shown that MmHm4R blocks MP5 activity in vitro and releases active MP5 after enzymatic cleavage.

[0139] Specifically, Figure 18 A shows a schematic diagram of MmHm4R. Figure 18 B shows the enzymatic cleavage of MmHm4R detected by SDS-PAGE. Figure 18 C shows the results of the red blood cell lysis experiment.

[0140] Figure 19 Shown is a lysis experiment of tumor cells MC38 after 24 hours of incubation.

[0141] Figure 20 Shown is a lysis experiment of tumor cells RM-1 after 24 hours of incubation.

[0142] Figure 21 The hinge region was shown to reversibly mask the agonistic ability of α4-1BB.

[0143] Specifically, Figure 21 A shows 4-1BB affinity detection. Figure 21 B shows that FcγR affinity can enhance the activity of 4-1BB antibodies. Figure 21 C shows the design modification of MmHm4R.

[0144] Figure 22 We show that RGD-targeted affinity integrin enhances 4-1BB antibody activity in MMHm4R.

[0145] Figure 23 It was shown that MmHm4R reduces the toxic side effects of MP5 and α4-1BB in vivo.

[0146] Specifically, Figure 23 A shows the experimental steps for toxicity evaluation in mice. Figure 23 B shows the weight change curve of mice after drug injection. Figure 23 C shows the red blood cell count. Figure 23 D shows the changes in hemoglobin. Figure 23 E and Figure 23 F shows the changes of liver toxicity indicators ALT and AST, respectively.

[0147] Figure 24 It was shown that MmHm4R has better pharmacokinetic properties.

[0148] Specifically, Figure 24 A shows the pharmacokinetic experimental steps. Figure 24 B and Figure 24 C shows the plasma concentration-time curve of MmHm4R.

[0149] Figure 25 It showed that MmHm4R had better tissue distribution characteristics.

[0150] Figure 26 It was shown that MmHm4R exhibited potent antitumor effects in tumor models without causing toxicity.

[0151] Specifically, Figure 26 A shows the experimental steps of MC38 pharmacodynamics. Figure 26 B shows the tumor growth curve and tumor photography at the end point of the experiment, showing that the MmHm4R group has the best drug efficacy.

[0152] Figure 27 It was shown that MmHm4R does not cause toxicity in tumor models.

[0153] Specifically, in terms of security, Figure 27 A shows the results of the red blood cell count. Figure 27 B shows the results of the hemoglobin test. Figure 27 C shows the platelet test results.

[0154] Figure 28 It was shown that MmHm4R does not cause liver toxicity in tumor models.

[0155] Specifically, Figure 28 A and Figure 28 B shows the test results of ALT and AST, the liver toxicity indicators.

[0156] Figure 29 The affinity of the MmCD3xEGFR multifunctional antibody to the CD3 antigen before and after MMP cleavage is shown.

[0157] Specifically, Figure 29 (Left) shows a schematic diagram of the MmCD3xEGFR multifunctional antibody, Figure 29 (Right) Shows the affinity of CD3 antibody to antigen before and after enzyme cleavage. DETAILED DESCRIPTION

[0158] After extensive and in-depth research, the inventors unexpectedly discovered for the first time a toxin / antibody dual-blocking prodrug molecule with good specificity and effectiveness, low off-target rate and high safety. The prodrug molecule connects melittin to a functional protein or polypeptide via a connecting segment, and the connecting segment comprises a cleavable peptide chain and a hinge region that are sensitive to an enzyme that is highly expressed specifically at the lesion site. By cleverly designing the connecting segment, it is possible to simultaneously mask (or significantly inhibit) the activity of the antibody and melittin in non-lesion sites, forming a dual-blocking state; in the lesion site, the melittin in the prodrug molecule and the functional protein or polypeptide are both released and their respective activities are restored, thereby being in a dual-activated state. The present invention was completed on this basis.

[0159] Specifically, taking the prodrug molecule MuHuA (melite venom-uPA enzyme-specific cleavable peptide chain-IgG hinge region-uPA enzyme-specific cleavable peptide chain-atezolizumab) and the prodrug molecule MmHm4R (melite venom MP5 mutant-MMP enzyme-specific cleavable peptide chain-IgG hinge region-MMP enzyme-specific cleavable peptide chain-4-1BB antibody) as examples, the present invention provides a tumor-specific toxin / antibody dual-blocking prodrug molecule, which comprises the following polypeptide elements: melittin, an antibody, and a connecting fragment, wherein the melittin is connected to the end of the antibody VH / VL variable region through a connecting fragment, and the connecting fragment includes, from N-terminus to C-terminus, a cleavable peptide chain sensitive to a tumor-specific highly expressed enzyme, a hinge region, and a cleavable peptide chain sensitive to a tumor-specific highly expressed enzyme. In the peripheral tissue environment, the activity of the antibody and bee venom in the prodrug molecule is masked (or significantly inhibited), forming a double-blocking state; in the tumor microenvironment, the uPA enzyme in the tumor microenvironment will cut the cleavable peptide chain in the connecting fragment, thereby releasing the antibody and bee venom in the prodrug molecule and restoring their respective activities, thus being in a double-activated state.

[0160] the term

[0161] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0162] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0163] As used herein, the terms "comprises," "includes," and "comprising" are used interchangeably to encompass not only closed definitions but also semi-closed and open definitions. In other words, the terms encompass "consisting of," "consisting essentially of."

[0164] Where a numerical range is provided, it is understood that every intervening integer of that value, every tenth of each intervening integer of that value, between the upper and lower limits of that range, and any other intervening values ​​in the stated range are encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any expressly excluded limits in the stated range. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," and the like.

[0165] As used herein, "chemically synthesized melittin", "synthetic melittin", "Free mel", and "mel" are used interchangeably and all refer to melittin used directly without forming a fusion protein.

[0166] As used herein, the antigen of the antibody comprises an immune checkpoint, a co-stimulatory molecule, an immune receptor complex, a cytokine, or a tumor antigen, which means that the antibody specifically binds to an immune checkpoint, a co-stimulatory molecule, an immune receptor complex, a cytokine, or a tumor antigen.

[0167] As used herein, the enzyme in the body of the subject to be administered refers to the enzyme corresponding to the enzyme-specific cleavable peptide chain in the prodrug molecule.

[0168] As used herein, an enzyme that specifically cleaves a peptide chain refers to an enzyme that can specifically cleave the peptide chain. For example, an enzyme that specifically cleaves a peptide chain that is uPA-specific is referred to as uPA; an enzyme that specifically cleaves a peptide chain that is MMP-specific is referred to as MMP; and an enzyme that specifically cleaves a peptide chain that is AEP-specific is referred to as AEP.

[0169] As used herein, the terms "linker" and "coupling element" are used interchangeably and both refer to the coupling element in the prodrug molecule of the present invention, which is located between the first active component and the second active component to couple the first active component and the second active component together. The coupling element comprises an enzyme-specific cleavable peptide chain and / or a polypeptide sequence containing a hinge region. In a specific embodiment, the coupling element comprises the structure: enzyme-specific cleavable peptide chain-polypeptide sequence containing a hinge region-enzyme-specific cleavable peptide chain. In a specific embodiment, the coupling element is cleaved by an enzyme in the body of the administered subject at or around the lesion site, thereby releasing the first active component and the second active component with their respective activities from the prodrug molecule.

[0170] As used herein, the phrases "the first active component and the second active component are released and restored to activity" and "the first active component and the second active component are separated, and the first active component restores toxicity while the second active component restores activity" are used interchangeably. That is, restoration of activity of the active component includes restoration of toxicity of the first component and restoration of activity of the second component. In specific embodiments, restoration of activity includes partial or complete restoration of activity.

[0171] Prodrug molecules of the present invention

[0172] The present invention provides a tumor-specific melittin / antibody prodrug molecule, comprising the following polypeptide elements: melittin, an antibody, and a linker fragment, wherein the melittin is linked to the ends of the antibody VH / VL variable regions via the linker fragment, and the linker fragment comprises, from the N-terminus to the C-terminus, a cleavable peptide chain sensitive to the tumor-specific highly expressed uPA enzyme, an IgG1 hinge region, and a cleavable peptide chain sensitive to the tumor-specific highly expressed uPA enzyme. In the peripheral tissue environment, the activities of the antibody and melittin in the prodrug molecule are masked (or significantly inhibited), forming a dual-blocking state; in the tumor microenvironment, the uPA enzyme in the tumor microenvironment cleaves the cleavable peptide chain in the linker fragment, thereby releasing the antibody and melittin in the prodrug molecule and restoring their respective activities, thereby forming a dual-activated state.

[0173] The present invention uses the hemolytic peptide melittin to assist in killing tumor cells and improves safety by constructing a tumor-specific melittin / antibody dual-blocking prodrug. This strategy can reduce the activity of the drug in peripheral tissues and restore activity after specific enzymatic cleavage in tumor tissues, thereby improving safety and reducing immune-related adverse reactions (irAEs). This approach can theoretically be applied to all antibodies targeting tumor antigens or immune cells. Here, antibodies targeting PD-L1 and 4-1BB are used as examples to illustrate.

[0174] Because PD-L1 antibodies are prone to triggering irAEs, which limits their use in patients with autoimmune diseases, the present invention constructed a melittin / PD-L1 antibody dual-blocking prodrug MuHuA, which connects melittin and the hinge region of human IgG1 to the N-terminus of the PD-L1-targeting atezolizumab (Atz) through a cleavable peptide chain that is sensitive to the tumor-specific and highly expressed uPA enzyme, thereby blocking the activity of melittin and Atz (M in MuHuA represents melittin, u represents the uPA enzyme-cleaved peptide chain, H represents the IgG1 hinge region, and A represents Atz).

[0175] In addition to significantly reducing the hemolytic toxicity of melittin and the autoimmune toxicity of Atz, MuHuA also exhibited favorable pharmacokinetic properties and higher tumor uptake. After enzymatic cleavage in the tumor microenvironment, MuHuA restored activity by releasing melittin and Atz, synergistically inhibiting tumor growth. The dual-blocking prodrug MuHuA could preferentially restore the viability of tumor-infiltrating lymphocytes and treat tumors without causing severe hemolysis or irAEs.

[0176] The clinical application of immune agonists such as 4-1BB antibody (abbreviated as α4) is severely limited by the systemic immune-related toxicity caused by their non-targeted effects. Using the technical solution of the prodrug molecule of the present invention, the present invention constructs a tumor-specific melittin mutant MP5 / anti-4-1BB dual-blocking prodrug (MmHm4), and further fuses the RGD polypeptide (targeted to bind to tumor tissue integrin) on the basis of MmHm4 to increase the targeting to tumor tissue, thereby obtaining MmHm4R (M in MmHm4R represents the melittin mutant MP5, m represents the MMP enzyme-cleaved peptide chain, H represents the IgG1 hinge region, 4 represents the 4-1BB antibody, and R represents the RGD amino acid sequence). MmHm4R connects the hemolytic peptide MP5 and the human IgG1 hinge region to the N-terminus of the 4-1BB agonist antibody through a peptide chain that can be cleaved by the MMP enzyme that is highly expressed in tumors, and at the same time fuses the integrin-targeted RGD peptide to the C-terminus of the Fc part. This design successfully masks the activity of MP5 and α4 in the peripheral circulation, significantly reducing the hemolytic effect of MP5 and the autoimmune toxicity of α4, while achieving better pharmacokinetic properties and higher tumor uptake. After cleavage in the tumor microenvironment, MmHm4R will restore activity by releasing MP5, which can induce tumor cell lysis, increase antigen exposure, and upregulate 4-1BB expression, while α4 will enhance intratumoral lymphocytes, thereby synergistically inhibiting tumor growth in both hot and cold tumor models.

[0177] The technical solution of the dual-blocking prodrug of the present invention can effectively solve the problem of non-targeted toxicity of antibodies, and can exert the different anti-tumor mechanisms of toxins and antibodies to achieve a synergistic therapeutic effect, while improving efficacy and safety.

[0178] The main advantages of the present invention include:

[0179] (a) The present invention provides a tumor-specific toxin / antibody dual-blocking prodrug molecule. In the peripheral tissue environment, the activities of the antibody and melittin in the prodrug molecule are masked (or significantly inhibited), forming a dual-blocking state. In the tumor microenvironment, the uPA enzyme in the tumor microenvironment cleaves the cleavable peptide chain in the linker fragment, thereby releasing both the antibody and melittin in the prodrug molecule and restoring their respective activities, thereby forming a dual-activated state.

[0180] (b) The tumor-specific toxin / antibody dual-blocking prodrug molecule of the present invention can reduce the activity of toxins and antibodies in peripheral tissues and restore their activity after specific enzymatic cleavage in tumor tissues, thereby effectively solving the problem of non-target toxicity of antibodies and being able to exert the different anti-tumor mechanisms of toxins and antibodies to achieve a synergistic therapeutic effect while improving the efficacy.

[0181] (c) The toxin / antibody dual-blocking prodrug molecules provided by the present invention can not only mask the activity of the antibody in peripheral tissues, significantly reduce the autoimmune toxicity of the antibody, and reduce immune-related adverse reactions, but also significantly mask the toxin and almost inhibit the hemolytic toxicity of the toxin (or its mutant), thereby showing better safety.

[0182] (d) Specifically, the present invention provides a melittin / PD-L1 antibody dual blocking prodrug (MuHuA) and a melittin mutant MP5 / anti-4-1BB dual blocking prodrug (MmHm4R), both of which show good pharmacokinetic properties and higher tumor uptake. After being cleaved by enzymes in the tumor microenvironment, both MuHuA and MmHm4R can restore activity by releasing melittin (or its mutants) and antibodies, thereby synergistically inhibiting tumor growth.

[0183] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0184] Example 1: MuHuA blocks the activity of melittin in vitro and releases active melittin after cleavage.

[0185] like Figure 1 As shown in A, in MuHuA, bee venom peptide, the hinge region of human IgG1 antibody, and two uPA cleavage linker peptides are fused to the N-termini of the heavy chain and light chain of atezolizumab (Atz). The hinge region is used to block the affinity of Atz for the PD-L1 antigen. At the same time, the hinge region and Atz can mask the activity of bee venom.

[0186] The SDS-PAGE results of MuHuA and MHA are as follows Figure 1As shown in B, the results show that, for the MuHuA of the present invention, after incubation with the uPA enzyme, melittin and Atz can be released from MuHuA; however, for the MHA control without the uPA cleavage linker (the linker between melittin and the hinge region, and between the hinge region and Atz does not contain the uPA cleavage linker), neither melittin nor Atz can be released.

[0187] To verify that the hemolytic activity of melittin in MuHuA could be masked, hemolysis experiments were performed using mouse red blood cells (RBCs). Figure 1 C and Figure 3 The results showed that melittin (Free Mel group) had hemolytic activity, while the hemolytic activity of melittin in the MuHuA of the present invention (Mel locked group) was blocked. When uPA was used to simulate MuHuA in the tumor microenvironment (TME) (Mel released group), it was observed that after uPA cleavage, melittin was effectively released from MuHuA, and melittin regained its hemolytic activity.

[0188] To further explore the activity of MuHuA against various tumor cells in vitro, 1000 MC38 cells were seeded in a 96-well plate and cultured for 12 hours to enter the logarithmic growth phase, and then incubated with different concentrations of free bee venom peptide (Free mel group), MuHuA (Mel locked group) or cleaved MuHuA (Mel released group). After incubation for 8 hours, 10 μL of CCK-8 reagent was added to each well and incubated for another 2 hours. The absorbance was measured at a wavelength of 450 nm using a microplate reader (reference wavelength 630 nm). Three replicate wells were set up for all experiments. In in vitro experiments, the concentration and incubation time of tumor cells were not sufficient for tumor cells to secrete enough uPA enzyme.

[0189] The results are as follows Figure 2 and Figure 4 shown.

[0190] The results showed that the MuHuA of the present invention (Mel locked group) had no killing effect on a variety of tumors, including mouse-derived MC38 and human-derived HT29 cells; and when uPA was enzymatically cleaved, bee venom peptide was effectively released from MuHuA, thereby effectively dissolving tumor cells, and the effect was comparable to that of directly synthesized bee venom peptide (Free mel group).

[0191] To verify the masking effect of MuHuA on the toxicity of melittin in vivo, C57BL / 6 mice were injected with PBS, 4 nmol melittin or 1 nmol MuHuA (containing 4 nmol melittin) via tail vein injection. The body weight of the mice was recorded and the Figure 5A's design underwent routine blood tests and chemistry analysis.

[0192] The results showed that compared with the PBS group, the synthetic melittin (Mel group) caused a sharp decrease in the body weight of mice ( Figure 5 B) and red blood cell count ( Figure 5 C) and hemoglobin ( Figure 6 , left) decreased significantly, platelets ( Figure 6 , right), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ( Figure 5 D) significantly increased. In contrast, there was no significant difference between the MuHuA group and the PBS group. This demonstrates that the MuHuA of the present invention almost completely masks the activity of melittin in lysing erythrocytes.

[0193] like Figure 5 The experimental design shown in E further explored the toxicity of higher doses of melittin and MuHuA (each containing 12 nM melittin) and monitored the corresponding mouse survival.

[0194] The results are as follows Figure 5 As shown in F, 100% mortality was observed within 2 days in mice treated with chemically synthesized melittin, whereas no mice treated with MuHuA died.

[0195] The results of this example show that the hemolytic toxicity of MuHuA in a normal healthy environment is greatly reduced, thereby increasing its clinical application potential; after being cleaved by the uPA enzyme in the tumor microenvironment, its activity is restored, and it can be used to inhibit tumors efficiently and safely.

[0196] Example 2: MuHuA reversibly masks the PD-L1 binding ability of atezolizumab, preventing autoimmune disease caused by PD-1 / PD-L1 blockade in NOD mice.

[0197] To evaluate the masking effect of the hinge region, the PD-L1 binding ability of atezolizumab and MuHuA was compared by ELISA. The specific method was as follows: 96-well plates were coated with 1.0 μg / mL mouse PD-L1 protein solution at 4°C overnight, then blocked with PBS containing 2% bovine serum albumin (BSA), and incubated with different concentrations of atezolizumab, HuA, MuHuA, or enzyme-cleaved MuHuA (Atz was released and restored to activity) at 37°C for 1 hour. Unbound histones were washed to remove the unbound histones, and then incubated with HRP-labeled goat anti-human IgG secondary antibody at 37°C for 1 hour. After washing, TMB single-component substrate solution was added for color development. After terminating the reaction with 2M sulfuric acid, the absorbance value at 450 nm (reference wavelength 630 nm) was read using a microplate reader (Teacan Infinite 200PRO).

[0198] The results are as follows Figure 7 As shown in the figures, the half-maximal effective concentrations (EC50) of atezolizumab (Ate group) and MuHuA (Atelockedin MuHuA group) were 0.08 nM and 106.31 nM, respectively, indicating that the PD-L1 binding ability of MuHuA was approximately 1000-fold weaker than that of atezolizumab.

[0199] The binding properties of MuHuA after uPA cleavage (Ate-released group) were similar to those of atezolizumab, indicating that the affinity for PD-L1 was almost fully restored after removal of the mask.

[0200] In addition, the similar PD-L1 binding affinity between HuA (Ate locked in HuA group, which does not contain melittin but only contains the hinge region, a cleavable linker peptide that can cleave uPA, and Atz) and MuHuA suggests that the hinge region can block the effect of the monoclonal antibody and melittin improves this blocking effect.

[0201] Non-obese diabetic (NOD) mice are genetically predisposed to spontaneously develop T cell-mediated type 1 diabetes and are therefore a common model for studying autoimmunity. Nine-week-old female NOD mice were intravenously administered with PBS, atezolizumab (400 pmol), or MuHuA (400 pmol). Blood glucose levels were monitored daily using glucose test strips, and mice with blood glucose levels ≥13.88 mmol / L were considered diabetic.

[0202] The results are as follows Figure 8 As shown, early-onset diabetes was observed in all mice treated with atezolizumab, but not in mice treated with MuHuA or PBS.

[0203] In addition to excessive thirst and polyuria, pancreatic tissue was dissected and RNA was extracted. Reverse transcription and qPCR were performed, and it was found that the mice in the atezolizumab group showed increased levels of pancreatic inflammatory cytokines ( Figure 9 ), which was not observed in the MuHuA or PBS groups. This model demonstrates that the activity of Atz in MuHuA is effectively masked in peripheral tissues, successfully avoiding the induction of lymphocyte activation or exacerbation of autoimmune diseases, thus opening the possibility of using anti-PD-L1 antibodies in patients with autoimmune diseases.

[0204] Example 3: MuHuA has better pharmacokinetic properties and tissue distribution.

[0205] To determine whether the masking effect improves the basic properties of MuHuA, including pharmacokinetic properties and tissue biodistribution, C57BL / 6 mice were given human IgG (hIgG), atezolizumab, or MuHuA (2 mg / kg) by intravascular injection. Blood was collected at different time points, and the plasma concentration of the injected protein was measured by ELISA.

[0206] The results are as follows Figure 10 As shown, although the elimination half-life (T1 / 2β) observed in the three groups was comparable, the plasma concentration of atezolizumab decreased more steeply than that of hIgG and MuHuA, with a shorter distribution half-life (T1 / 2α) and a lower area under the curve (AUC).

[0207] We further investigated the tissue distribution of atezolizumab or MuHuA (0.5 mg / kg) by injecting C57BL / 6 mice bearing RM-1 tumors and collecting major organs and tumors 6 days after injection.

[0208] ELISA results Figure 11 As shown, the results showed that MuHuA was expressed at lower levels in lymphoid organs (including spleen, mesenteric lymph nodes, and thymus) compared with atezolizumab ( Figure 11 , left panel), with higher levels in the blood and blood-rich tissues (such as the heart, lungs, and liver), consistent with the pharmacokinetic results.

[0209] In addition, the concentration of MuHuA in the tumor was significantly increased compared with the control, reaching 3 times that of Atz injection alone ( Figure 11 , right panel). MuHuA in tumors released active antibody at levels comparable to atezolizumab alone, whereas released atezolizumab was virtually undetectable in normal organs (0.7 ng / g in the liver, 0.3 ng / g in the kidney, and none in the blood, heart, spleen, lung, mesenteric lymph nodes, or thymus).

[0210] In summary, the results of this example indicate that the masking strategy of MuHuA weakens its uptake by lymphoid organs with high PD-L1 expression and promotes its preferential accumulation and activation within the TME.

[0211] Example 4: MuHuA exhibits potent anti-tumor effects in tumor models without causing toxicity.

[0212] like Figure 12 The technical scheme shown in A studied the anti-tumor effect of MuHuA in the MC38 mouse colon cancer model.

[0213] The results showed that atezolizumab showed 31% tumor growth inhibition (TGI), but unexpectedly, melittin (used alone or with atezolizumab) promoted tumor growth ( Figure 12 B). In contrast, equimolar MuHuA (350 pmol, or 3 mg / kg) exhibited more potent antitumor activity (70% TGI) and longer survival ( Figure 12 C), which was not observed in the MHA group, demonstrating that this effect was mediated through protease-dependent release.

[0214] Flow cytometry (FCM) was used to detect immune activation in the tumor. The results showed that after MuHuA treatment, the T cells in the tumor ( Figure 13 ), CD8+ T cells ( Figure 14 ) and NK cells ( Figure 15 ) increased significantly, indicating that it significantly improved the tumor microenvironment and promoted the activation of immune cells.

[0215] The mice in the MuHuA group showed comparable blood test results to those in the hIgG group, whereas free melittin Mel resulted in Figure 16 ) decreased, hemoglobin ( Figure 17 Left) decreased and platelets increased ( Figure 17 Right). This indicates that MuHuA significantly reduces the toxicity of melittin in healthy tissues.

[0216] The in vivo experiments in this example demonstrate that MuHuA can stimulate tumor-specific immunity while avoiding systemic toxicity caused by hemolysis.

[0217] Example 5: MmHm4R blocks MP5 activity in vitro and releases active MP5 after enzymatic cleavage.

[0218] In order to address the immune-related side effects of 4-1BB antibodies, an anti-4-1BB agonist (α4) / melittin mutant (melittin P5, MP5) dual-blocking prodrug MmHm4R was developed to retain its anti-tumor activity while limiting systemic toxicity. The hinge region of MmHm4R will block the antigen binding of α4, while the hinge region and α4 will block the activity of MP5. At the same time, the RGD peptide targeting integrin is fused to the C-terminus of the heavy chain to enhance direct targeting ability. Specifically, Figure 18 As shown in A.

[0219] SDS-PAGE analysis Figure 18 As shown in Figure B, after incubation with MMP2, MP5 and α4 can be released from MmHm4R, but the non-cleavable MH4R (the linker between MP5, hinge region, and α4 is a non-cleavable linker) in which the cleavage linker peptide is deleted cannot be released.

[0220] like Figure 18As shown in C, direct use of chemically synthesized MP5 (MP5 group) caused significant erythrocyte lysis in the hemolysis experiment, while the lytic activity in MmHm4R was completely inhibited.

[0221] like Figure 19 and Figure 20 As shown in Figure 2, the MMP2 was cleaved and released from the MmHm4R fusion construct. The proteins before and after cleavage were incubated in cells for 24 hours. The results showed that the released MP5 MP5 could effectively kill a variety of tumor cells, including mouse MC38 cells ( Figure 19 ) and mouse RM-1 cells ( Figure 20 ), and its cytolytic activity was comparable to that of chemically synthesized free MP5.

[0222] Example 6: Hinge region and melittin reversibly mask the agonistic ability of α4-1BB.

[0223] The affinity of MmHm4R for 4-1BB protein was detected by ELISA. The 96-well plate was coated with 5.0 μg / mL mouse 4-1BB protein solution at 4°C overnight, blocked the next day, and incubated with different concentrations of target protein at 37°C for 1 hour. The unbound target protein was washed to remove the unbound target protein, and then incubated with HRP-labeled goat anti-human IgG secondary antibody at 37°C for 1 hour. After washing, TMB single-component substrate solution was added for color development. After terminating the reaction with 2M sulfuric acid, the absorbance value at 450nm (reference wavelength 630nm) was read on a microplate reader (Teacan Infinite 200 PRO). The results are as follows Figure 21 As shown in A, the binding ability of MmHm4R to mouse 4-1BB was weakened by more than 400 times due to masking by the hinge region and melittin, but was restored after enzymatic cleavage.

[0224] like Figure 21 As shown in Figure B, the co-stimulatory effect of α4 was demonstrated in an in vitro experiment using spleen cells isolated from mice. Since the function of anti-4-BB antibodies is greatly enhanced under cross-linking conditions, when α4 co-exists with RAW264.7 cells, a mouse macrophage cell line that can express Fcγ receptors, the binding of the Fc segment of α4 to FcγR promotes the formation of a multivalent cross-linked structure, thereby observing an enhancement of α4 function and an increase in INF-γ secretion levels.

[0225] To address hepatotoxicity mediated by Fcγ receptor-expressing liver sinusoidal endothelial cells and Kupffer cells, the Fc of α4 was engineered and an RGD peptide was fused to its C-terminus, such as Figure 21 As shown in C. The binding of Fc to FcγR is completely blocked after modification, while the fused RGD peptide can mediate the binding of integrins expressed by tumors.

[0226] In vitro costimulation experiments were performed using the modified constructs, and the results were as follows Figure 22 As shown, MMP2-cleaved MmHm4R exhibited a costimulatory effect, whereas uncleaved MmHm4R or MMP2-cleaved MH4R showed no costimulatory effect.

[0227] The costimulatory effect of MMP2-cleaved MmHm4R, but not α4 or MmHm4 (which lacks the RGD sequence compared to MmHm4R), can be enhanced by integrin-expressing MC38 cells. This is because integrins on the MC38 surface bind to RGD, promoting cross-linking of the α4 antibody released after enzymatic cleavage, thereby enhancing the costimulatory effect. All of these data indicate that the modification of the present invention successfully shifts the costimulatory effect of α4 from an FcγR-dependent mechanism to one that relies on tumor-specific integrins.

[0228] Example 7: MmHm4R reduces the toxic side effects of MP5 and α4-1BB in vivo.

[0229] To verify the improvement of the safety of MP5 and α4-1BB by MmHm4R in animals, PBS, 4 nmol MP5, 1 nmol α4, or 1 nmol MmHm4R (containing 4 nmol MP5 and 1 nM α4) were injected into the tail vein of C57BL / 6 mice, and the body weight of the mice was continuously recorded. The experimental protocol was as follows: Figure 23 As shown in A, body and organ weights were measured at the end of the experiment, and routine blood tests and chemical analyses were performed.

[0230] The mouse body weight was measured as follows Figure 23 As shown in B, the results showed that the body weight of mice in the MP5 group decreased sharply, while there was no significant difference in body weight between the MmHm4R group and the PBS group.

[0231] Blood test results are as follows Figure 23 C. Figure 23 D. Figure 23 E and Figure 23 As shown in F.

[0232] The results showed that a significant decrease in red blood cell count and hemoglobin, as well as an increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected in the MP5 group, but not in the MmHm4R group. In addition, the injection of α4 caused a significant increase in ALT and AST, which were not observed in the MmHm4R group.

[0233] The results of this example indicate that MmHm4R can effectively prevent the host from hemolysis caused by systemic administration of MP5 and the immune-related liver damage side effects caused by α4.

[0234] Example 8: MmHm4R has better pharmacokinetic properties and tissue distribution.

[0235] In order to examine the pharmacokinetic properties and tissue biodistribution of MmHm4R, the Figure 24 Experimental protocol shown in A: C57BL / 6 mice were intravenously injected with human IgG (hIgG), α4, or MmHm4R (2 mg / kg), and the plasma concentrations of the injected proteins were measured by ELISA at different time points.

[0236] The drug-dose curves of IgG, α4 and MmHm4R are as follows Figure 24 B and Figure 24 As shown in Figure 5C, the results indicate that, unlike α4, which exhibits faster distribution and metabolic clearance, MmHm4R displays pharmacokinetic properties similar to hIgG.

[0237] C57BL / 6 mice inoculated with B16F10 tumors were further injected with α4 or MmHm4R (1 mg / kg), and tissues were collected 6 days after injection to investigate the tissue distribution of MmHm4R.

[0238] The ELISA results of α4, MmHm4dR, MmHm4R and Releasedα4from MmHm4Rde in each tissue are as follows Figure 25 The results showed that, compared with hIgG, α4 and MmHm4R levels in normal organs were comparable, indicating that α4 has limited distribution in normal tissues. Compared with α4 and MmHm4, MmHm4R levels in tumor tissues were elevated, approximately fourfold higher than in the control group, confirming the targeting effect of the RGD peptide. Furthermore, nearly all MmHm4R in tumors was cleaved into active α4, while the cleavage rate in normal tissues was lower.

[0239] In summary, the results of this example indicate that the masking strategy of MmHm4R prolongs its circulation time in vivo and promotes its preferential accumulation and efficient activation in tumor tissues.

[0240] Example 9: MmHm4R exhibits potent anti-tumor effects in tumor models without causing toxicity.

[0241] according to Figure 26 The pharmacodynamic experimental steps of A were used to study the anti-tumor effect of MmHm4R in the MC38 mouse colon cancer model.

[0242] Tumor growth curve and corresponding tumor photos are shown in Figure 2. Figure 26 As shown in B.

[0243] A single treatment with α4 showed 48% tumor growth inhibition (TGI). However, both the MP5 and MP5+α4 groups unexpectedly promoted tumor growth. The MH4R group had no antitumor effect, while an equimolar dose of MmHm4R (350 pmol, or 3 mg / kg) exhibited significantly more pronounced antitumor activity (92% TGI) and longer survival than the MmHm4 group (79% TGI), demonstrating that its antitumor effect was dependent on enzymatic cleavage-mediated release and could be enhanced by the RGD peptide.

[0244] Routine blood tests such as Figure 27 、 28 The results showed that the red blood cells of MP5 group and α4 group ( Figure 27 A) Lower hemoglobin Figure 27 B), high platelet count ( Figure 27 C); plasma alanine aminotransferase (ALT) ( Figure 28 A) and aspartate aminotransferase (AST) ( Figure 28 B) The concentration was higher. However, this phenomenon was not observed in the MmHm4 and MmHm4R groups.

[0245] The data of this example show that MmHm4R can specifically activate tumor immunity and avoid systemic toxicity.

[0246] Example 10 Hinge region and melittin reversibly mask the affinity of CD3

[0247] In order to reduce the side effects of excessive immune activation caused by CD3 antibody in CD3xEGFR bispecific antibody, MP5 and hinge regions were used to mask its activity, and MmCD3xEGFR trifunctional prodrug molecule ( Figure 29 (left). In the MmCD3xEGFR molecule, the MP5 and hinge region block the activity of the CD3 antibody. The hinge region and the CD3 antibody simultaneously block the activity of MP5, reducing the toxicity of MP5 and CD3 antibodies in peripheral tissues. Upon co-incubation with MMP2, MP5 is released from the MmCD3xEGFR, restoring cell lysis and restoring the affinity of the CD3 antibody.

[0248] The affinity of the CD3 antibody after masking and enzyme cleavage was determined by ELISA, with a CD3 monoclonal antibody used as a control. Specifically, a 96-well plate was coated with a 2.0 μg / mL mouse CD3 protein solution at 4°C overnight, then blocked with PBS containing 2% bovine serum albumin (BSA). Plates were incubated with various concentrations of αCD3 (CD3 monoclonal antibody), MmCD3xEGFR, or enzyme-cleaved MmCD3xEGFR for 1 hour at 37°C. Unbound proteins were removed by washing, and the plates were then incubated with an HRP-labeled goat anti-human IgG secondary antibody at 37°C for 1 hour. Unbound secondary antibody was removed by washing, and TMB single-component substrate solution was added for color development. The reaction was terminated with 2M sulfuric acid, and the absorbance at 450 nm (reference wavelength 630 nm) was read using a microplate reader (Teacan Infinite 200PRO).

[0249] The results showed that the affinity of CD3 monoclonal antibody was 28.64nM, and the affinity of CD3 antibody in MmCD3xEGFR was masked by about 560 times, about 1135nM; after MMP2 enzyme cleavage, MmCD3xEGFR was unmasked and the affinity for CD3 protein was restored to 34.41nM, which was no significant difference from CD3 monoclonal antibody ( Figure 29 , right). This experiment demonstrates that a dual-blocking approach can reversibly mask the activity of CD3 monoclonal antibodies, reducing systemic toxic side effects and increasing anti-tumor dosage and efficacy in vivo.

[0250] discuss

[0251] On the one hand, when in non-lesion sites, the dual-blocking prodrug molecule of the present invention is in an active blocking state, and the activity of the active component in the prodrug molecule is significantly masked or completely masked, thereby minimizing the toxic and side effects of the active component on non-lesion healthy tissues.

[0252] On the other hand, after reaching the lesion site, the active component in the prodrug molecule of the present invention is released and its activity is restored, so that the active component reaches a higher drug concentration at the lesion site, thereby reducing the dosage of the active component, more effectively exerting the therapeutic effect, and providing more targeted treatment for the disease. In addition, the synergistic anti-tumor effect of the two components in the prodrug molecule can be exerted to improve the therapeutic effect, thereby minimizing the nonspecific effects and off-target effects of the active component, and retaining the specificity and effectiveness of the active component in precisely acting on diseased cells or tissues to the greatest extent.

[0253] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A dual-blocking prodrug molecule, characterized in that: The prodrug molecule comprises: (a) a first active component, (b) a second active component, and (c) a coupling element located between the first active component and the second active component for coupling the first active component and the second active component together; wherein the first active component is a toxin molecule and the second active component is a protein with a predetermined activity; Furthermore, the prodrug molecule has a first state. When the prodrug molecule is in the first state (or blocking state), the first active component is in a toxicity blocking state, and the activity of the second active component is blocked by the first active component and / or the coupling element. Furthermore, when the prodrug molecule is cleaved, it is converted into a drug having a second state; in the second state, the first active component and the second active component are separated, and the first active component regains its toxicity while the second active component regains its activity.

2. The prodrug molecule according to claim 1, characterized in that The dual-blocking prodrug molecule is cleaved at or around the lesion site.

3. The prodrug molecule according to claim 1, characterized in that The coupling element comprises a structure shown in Formula II: Z21-Z22-Z23(II) Wherein, the Z21 is an enzyme-specific cleavable peptide chain; Z22 is a polypeptide sequence containing a hinge region; and The Z23 is an enzyme-specific cleavable peptide chain.

4. The prodrug molecule according to claim 3, characterized in that When the enzyme corresponding to the enzyme-specific cleavable peptide chain is absent, the toxicity of the toxin is masked, and the activity of the functional polypeptide or protein is masked; When the enzyme corresponding to the enzyme-specific cleavable peptide chain is present, the toxin is released and its toxicity is restored, and / or the functional polypeptide or protein is released and its activity is restored.

5. A recombinant protein or polypeptide, characterized in that The recombinant protein or polypeptide comprises: (i) the prodrug molecule according to claim 1; (ii) Optional functional polypeptide or protein.

6. A polynucleotide, characterized in that The polynucleotide encodes the prodrug molecule according to any one of claims 1 to 4, or the recombinant protein or polypeptide according to claim 5.

7. A carrier, characterized in that The vector comprises the polynucleotide of claim 6.

8. A host cell, characterized in that The host cell comprises the vector of claim 7 or the polynucleotide of claim 6 is integrated into its genome.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the prodrug molecule according to claim 1.

10. Use of the prodrug molecule according to claim 1, the recombinant protein or polypeptide according to claim 5, the polynucleotide according to claim 6, the vector according to claim 7, the host cell according to claim 8, or the pharmaceutical composition according to claim 9, characterized in that: Used for preparing a medicine kit for treating tumors.

11. A medicine box, characterized in that: The drug kit comprises the prodrug molecule of claim 1, the recombinant protein or polypeptide of claim 5, the polynucleotide of claim 6, the vector of claim 7, the host cell of claim 8, or the pharmaceutical composition of claim 9.

12. The method for activating a prodrug molecule according to claim 1, characterized in that: The following steps are involved: (S1) providing the prodrug molecule according to claim 1; (S2) activating the prodrug molecule according to claim 1 in the presence of an enzyme corresponding to the enzyme-specific cleavable peptide chain, thereby producing a toxin and / or an antibody.

13. A reaction system for in vitro activation of the prodrug molecule according to claim 1, characterized in that: The reaction system comprises: (i) the prodrug molecule according to claim 1; and (ii) a sample comprising an enzyme corresponding to the enzyme-specific cleavable peptide chain in the prodrug molecule of claim 1.

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