Fusion protein taking peptide-N-glycosidase as active component as well as preparation method and application of fusion protein

Abstract

The invention discloses a fusion protein taking peptide-N-glycosidase as an active component as well as a preparation method and application of the fusion protein, and belongs to the technical field of biological medicines. The fusion protein comprises: (a) peptide-N-glycosidase or a catalytically active fragment thereof; (b) an immunoglobulin Fc domain, or a combination of a tumor or immune cell antigen binding domain and an immunoglobulin Fc domain; (c) a linker peptide; wherein the form of the tumor or immune cell antigen binding domain is Fab, scFv or VHH; the peptide-N-glycosidase or the catalytic activity fragment of the peptide-N-glycosidase is connected with the Fc structural domain of the immunoglobulin through the connecting peptide; the immunoglobulin Fc domain mediates the fusion protein to form a homodimer or a heterodimer. According to the invention, the synergistic function of targeted binding and local deglycosylation of the target molecule is realized, so that the immunosuppressive activity of the target molecule is interfered, and the anti-tumor immune response is enhanced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a fusion protein with peptide-N-glycosidase as the active ingredient, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The immune escape mechanism of malignant tumors is closely related to abnormal protein glycosylation modification. Studies have shown that more than 50% of solid tumor cell surface proteins exhibit excessive N-glycosylation. These abnormal glycans participate in regulating the function and stability of immune checkpoints such as PD-1 / PD-L1 by forming a "glycosylation barrier," becoming a key mechanism for tumors to evade host immune attack.

[0004] Immune checkpoint blockade (ICB) therapy, which restores the tumor-specific killing function of T lymphocytes by inhibiting the binding of immune checkpoint molecules such as PD-1 / PD-L1, has made significant progress in the treatment of more than 20 types of cancer, including melanoma and non-small cell lung cancer. However, this therapy still faces challenges such as low overall efficacy as a single agent and the easy occurrence of acquired resistance, and there is an urgent need to develop differentiated strategies.

[0005] The glycosylation status of immune checkpoint molecules is a crucial factor influencing treatment efficacy. Taking PD-L1 as an example, multiple N-glycosylation sites in its extracellular domain (such as Asn192, Asn200, and Asn219) maintain protein stability, enhance its binding affinity to PD-1, promote T cell exhaustion, and lead to tumor immune escape. Tumor cells can further enhance the glycosylation level of PD-L1 by upregulating glycosyltransferases (such as TGF-β-induced B3GNT3), exacerbating immunosuppression. Similarly, the branching degree of the N-glycans in CTLA-4 affects its residence time on the T cell surface, while the glycosylation status of TIM-3 directly regulates its binding efficiency with its ligand Galectin-9; both participate in tumor immune escape through glycosylation modification.

[0006] While improved strategies targeting glycosylation are seen as a potential direction for overcoming immunotherapy resistance, existing methods have significant limitations: small molecule inhibitors of glycosyltransferases are mostly still in preclinical or early-stage trials due to broad-spectrum toxicity and off-target risks; human N-glycosyltransferase (NGLY1) is mainly located in the cytoplasm and cannot effectively act on key immune checkpoint proteins on the cell surface (such as PD-L1), thus its activity is also limited; and monotherapy is even more difficult to overcome efficacy bottlenecks. Therefore, developing new strategies that can specifically and efficiently intervene in the glycosylation state of immune checkpoints in the tumor microenvironment has become an urgent problem to be solved in the field of tumor immunotherapy. Summary of the Invention

[0007] In view of this, the present invention provides a fusion protein with peptide-N-glycosidase as the active ingredient, its preparation method, and its application. The fusion protein of the present invention achieves a synergistic function of targeted binding and local deglycosylation of the target molecule by coupling the tumor or immune cell antigen-binding domain with the peptide-N-glycosidase molecule, thereby interfering with the immunosuppressive activity of the target molecule and enhancing the anti-tumor immune response.

[0008] In a first aspect, the present invention provides a fusion protein with peptide-N-glycosidase as the active ingredient, comprising: (a) Peptide-N-glycosidase or its catalytically active fragment; (b) Immunoglobulin Fc domain, or a combination of tumor or immune cell antigen-binding domain and immunoglobulin Fc domain; (c) Linking peptides; The tumor or immune cell antigen-binding domain is in the form of Fab, scFv, or VHH; the peptide-N-glycosidase or its catalytically active fragment is linked to the immunoglobulin Fc domain via the linker peptide; the immunoglobulin Fc domain mediates the formation of homodimers or heterodimers of the fusion protein.

[0009] Secondly, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the aforementioned fusion protein.

[0010] Thirdly, the present invention provides a carrier comprising the above-mentioned nucleic acid molecules.

[0011] Fourthly, the present invention provides a host cell comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0012] Fifthly, the present invention provides a method for preparing the above-mentioned fusion protein, comprising the following steps: Construct an expression vector containing the gene encoding the fusion protein; Host cells containing the expression vector are constructed by transient or stable transfection of host cells; The fusion protein was isolated and purified from the host cell.

[0013] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the above-described fusion protein and a pharmaceutically acceptable carrier.

[0014] In a seventh aspect, the present invention provides the use of the above-described fusion protein or the above-described pharmaceutical composition, said use including one or more of the following: (i) Preparation of antitumor drugs; (ii) Preparation of antitumor drugs for use in combination with immune checkpoint inhibitors; (iii) Preparation of antitumor drugs for use in combination with T cell adoptive transfer; (iv) To prepare drugs for postoperative adjuvant immunotherapy to prevent tumor recurrence or metastasis after surgery; (v) To prepare combination therapy drugs to enhance the effects of radiotherapy or chemotherapy; (vi) Prepare pharmaceutical compositions for the treatment of patients with drug-resistant or recurrent tumors.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The fusion protein of the present invention achieves the synergistic function of specific targeting binding to target molecules and local efficient deglycosylation by coupling the tumor or immune cell antigen binding domain with the peptide-N-glycosidase molecule. This precisely solves the limitation of existing N-glycosylation enzymes that cannot effectively act on key immune checkpoint proteins on the cell surface, and significantly improves the pertinence and effectiveness of deglycosylation intervention.

[0016] (2) The introduction of the immunoglobulin Fc domain into the fusion protein of the present invention not only enhances molecular stability and target binding ability through dimerization, but also reduces off-target toxicity through gene mutation of the Fc segment (such as removal of ADCC effect), taking into account both functional activity and safety, and breaking through the clinical application limitations of traditional glycosyltransferase small molecule inhibitors due to broad-spectrum toxicity and off-target risk.

[0017] (3) The peptide-N-glycosidase (such as PNGase F) used in the fusion protein has the characteristics of high specificity and strong catalytic efficiency. Its synergistic effect with the target domain can effectively interfere with the glycosylation modification and immunosuppressive activity of the target molecule (such as PD-L1), thereby significantly enhancing the anti-tumor immune response and providing a new strategy for breaking through the efficacy bottleneck of monotherapy.

[0018] (4) Based on the key role of PD-L1 glycosylation modification in immune function regulation, and combined with the high specificity and efficient catalytic properties of PNGase F deglycosylation, this invention designs a fusion protein composed of an anti-PD-L1 antibody and PNGase F. This protein targets and binds to PD-L1 through antibody, while simultaneously utilizing PNGase F for local deglycosylation, synergistically interfering with the immunosuppressive activity of PD-L1 and enhancing the anti-tumor immune response; it verifies the feasibility of the "targeting antibody + peptide-N-glycosidase" fusion strategy in tumor immunotherapy and provides a technical path for developing fusion proteins of other glycosylated immunomodulatory molecules. Attached Figure Description

[0019] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0020] Figure 1 This describes the construction and purification of the αPD-L1-PNGase F fusion protein in Example 1 of this invention, where A is a schematic diagram of the structure and B is the SDS-PAGE band of the fusion protein; Figure 2 This refers to the antitumor effect of intratumoral injection of αPD-L1-PNGase F fusion protein, αPD-L1, and PNGase F-Fc in Example 2 of the present invention. Figure 3 This refers to the antitumor effect of intraperitoneal injection of equimolar doses of the PNGase F, PNGase F-Fc, and αPD-L1-PNGase F fusion protein in Example 2 of the present invention. Figure 4 This is a graph showing the relationship between the antitumor activity of the αPD-L1-PNGase F fusion protein and the dosage (0~75μg) in Example 3 of the present invention; Figure 5 This is a graph showing the relationship between the dose of αPD-L1-PNGase F fusion protein and body weight (0~450μg) in Example 3 of this invention; Figure 6 The antitumor efficacy of αPD-L1-PNGase F fusion protein, αPD-L1 monoclonal antibody, PNGase F, αPD-L1+PNGase F and enzyme activity mutant αPD-L1-PNGase F (αPD-L1-PNGase F-mut) in Example 4 of this invention; Figure 7This describes the antitumor effect of NSG immunodeficient mice in MC38 subcutaneous tumor inoculation and intratumoral injection of PNGase F-Fc fusion protein in Example 4 of this invention. Figure 8 This refers to the antitumor efficacy of αPD-L1-PNGase F in the MC38-PD-L1-KO subcutaneous tumor mouse model in Example 5 of this invention; Figure 9 This is the verification result of the anti-tumor effect of the αPD-L1-PNGase F fusion protein in Example 6 of the present invention depending on CD8+ T cells; Figure 10 The anti-tumor effect of the αPD-L1-PNGase F fusion protein in Example 7 of this invention depends on the verification results of CD8+ T cells already present in the tumor; Figure 11 This is the verification result of the antitumor effect of the αPD-L1-PNGase F fusion protein in Example 8 of the present invention being dependent on the secretion of IFN-γ; Figure 12 The αPD-L1-PNGase F fusion protein in Example 9 of this invention induces tumor-specific CD8. + T cells are increased, of which A is intratumoral CD8. + T cell percentage; B represents intratumoral CD8. + T cell count; C represents tumor-specific CD8 within the tumor. + T ratio; D is tumor-specific CD8 within the tumor. + T cell count; E is the CD8 / Treg ratio; F is the concentration of IFN-γ in CD8 cells. + The proportion of T cells. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] Generally, the terms and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of this invention are generally performed according to conventional methods known in the art.

[0023] Terminology Explanation To better understand this invention, the definitions and explanations of relevant terms are provided below.

[0024] The term "antibody" encompasses a variety of antibody structures, including polyclonal antibodies, monospecific antibodies, and multispecific antibodies (e.g., bispecific antibodies). A natural, intact antibody typically refers to a Y-shaped tetrameric protein consisting of two heavy chains (H) and two light chains (L) held together by covalent disulfide bonds and non-covalent interactions. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity-determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)).

[0025] The term "antigen-binding domain" refers to a functional region in an immune molecule (such as an antibody or its derivatives) responsible for specifically recognizing and binding to a target antigen. Its core function is to precisely bind to specific antigens (such as PD-L1 or CTLA-4 molecules on the surface of tumor cells or immune cells) through complementary intermolecular structures (such as spatial conformation and charge matching), thereby mediating subsequent immune regulation or targeting. In this invention, the antigen-binding domain can take the form of Fab (antigen-binding fragment), scFv (single-chain variable fragment), VHH (variable region of a nanobody), etc., and is a key functional module for fusion proteins to target molecules on the surface of tumor or immune cells.

[0026] The term "antigen-binding fragment" refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to an antigen that binds specifically to the full-length antibody, and / or competes with the full-length antibody for binding to the same antigen.

[0027] The term "Fc domain" refers to the region in an antibody molecule composed of heavy chain constant regions, located at the tail of a Y-shaped antibody. It contains the second (CH2) and third (CH3) constant regions of the first heavy chain, linked by disulfide bonds to the second and third constant regions of the second heavy chain. The Fc region may also contain all or part of the hinge region. Its primary function is to mediate the immune effects of the antibody, such as binding to Fc receptors on the surface of immune cells to trigger phagocytosis or cytotoxicity, and it can also participate in complement system activation. Simultaneously, it maintains the stability of the antibody molecule and affects its half-life in vivo. In fusion protein design, modifications to the Fc domain (such as gene mutations) can further modulate functional activity, reduce off-target toxicity, or optimize action properties.

[0028] The terms "anti-PD-L1 antibody," "anti-CTLA4 antibody," "anti-PD-1 antibody," and "anti-EGFR antibody" include antibodies or antigen-binding fragments that specifically recognize or bind to PD-L1, CTLA4, PD-1, and EGFR. The terms "anti-PD-L1 antibody," "antiPDL1," and "αPD-L1" are used interchangeably.

[0029] The term "fusion protein" refers to a polypeptide having two or more parts covalently linked together, each part being a peptide with different properties. These properties can be biological, such as in vitro or in vivo activity. They can also be simple chemical or physical properties, such as binding to a target antigen, catalysis of a reaction, etc. The two parts can be directly linked by a single peptide bond or by linking peptides containing one or more amino acid residues.

[0030] The term "PD-L1," also known as programmed death ligand 1, is a 40 kDa type 1 transmembrane protein that is presumed to play a major role in suppressing the adaptive immune system. PD-L1 is the major ligand of programmed death 1 (PD-1).

[0031] The term "ADCC effect" refers to antibody-dependent cell-mediated cytotoxicity, an immune defense mechanism. The process involves antibodies specifically binding to antigens on the surface of target cells (such as virus-infected cells or tumor cells) via their Fab fragment, while simultaneously binding to Fc receptors on the surface of effector cells (such as natural killer cells, NK cells, and macrophages). This activates effector cells to release toxic substances such as perforin and granzymes, ultimately leading to the rupture and death of target cells. This effect is an important mechanism of synergistic action between antibody-mediated adaptive immune responses and innate immune cells, playing a crucial role in antiviral infections and tumor immunotherapy.

[0032] The term "knob" refers to a "bump" structure introduced into the CH3 domain (the third domain of the heavy chain constant region) of an antibody through amino acid mutation. This is typically achieved by replacing a smaller amino acid residue (such as threonine T) with a larger residue (such as tryptophan W), creating a spatially "bumpy" structure.

[0033] The term "hole," the "depression" structure corresponding to the knockb, is formed by replacing a larger amino acid residue (such as tyrosine Y) with a smaller residue (such as serine S or alanine A) at the corresponding position of another antibody CH3 domain, and is spatially complementary to the knockb.

[0034] The term "Knob-in-Hole mutation strategy" is a protein engineering technique used for the assembly of bispecific antibodies. Its core principle is to introduce knock (convex) and hole (concave) mutations into the CH3 domains of two different antibody heavy chains, respectively. By utilizing spatial complementarity (convex-concave chimerism), the two heavy chains are forced to preferentially and correctly pair, avoiding misbinding of homologous chains.

[0035] The term "pharmaceuticalally acceptable" means that the carrier, diluent, excipient and / or salt thereof are chemically and / or physically compatible with other components in the formulation and are physiologically compatible with the recipient.

[0036] Fusion protein with peptide-N-glycosidase as the active ingredient This invention provides a fusion protein with peptide-N-glycosidase as the active ingredient, comprising: (a) Peptide-N-glycosidase or its catalytically active fragment; (b) Immunoglobulin Fc domain, or a combination of tumor or immune cell antigen-binding domain and immunoglobulin Fc domain; (c) Linking peptides; The tumor or immune cell antigen-binding domain is in the form of Fab, scFv, or VHH; the peptide-N-glycosidase or its catalytically active fragment is linked to the immunoglobulin Fc domain via the linker peptide; the immunoglobulin Fc domain mediates the formation of homodimers or heterodimers of the fusion protein.

[0037] The fusion protein of this invention achieves a synergistic function of specific targeting and efficient local deglycosylation of target molecules by coupling the tumor or immune cell antigen-binding domain to a peptide-N-glycosidase molecule. In the absence of the tumor or immune cell antigen-binding domain, the immunoglobulin Fc domain can be directly linked to the peptide-N-glycosidase or its catalytically active fragment via a linker peptide.

[0038] In this invention, the peptide-N-glycosidase is selected from PNGase F, PNGase F-II, and PNGase H. + The peptide-N-glycosidase may contain one or more of PNGase Rc, PNGase A, Endo H, Endo F3, Endo S, α-fucosidase, or NGLY1, preferably PNGase F. Further, the amino acid sequence of the peptide-N-glycosidase is as shown in SEQ ID NO. 1, or a variant having at least 80% of the same amino acid sequence as SEQ ID NO. 1 and retaining peptide-N-glycosidase activity.

[0039] In this invention, the tumor or immune cell antigen-binding domain specifically binds to one or more of the following targets: PD-L1, CTLA-4, PD-1, TIGIT, TIM3, LAG3, 41BB, ICOS, GITR, 2B4, B7H3, B7H4, EGFR, CLDN18.2, HER2, Trop2, or Nectin-4, preferably PD-L1, CTLA-4, PD-1, or EGFR, and more preferably PD-L1.

[0040] In this invention, the immunoglobulin Fc region is natural immunoglobulin Fc or immunoglobulin Fc with ADCC effect removed through gene mutation. Preferably, the immunoglobulin Fc is human immunoglobulin IgG Fc. Its amino acid sequence is shown in SEQ ID NO.2, SEQ ID NO.3 (ADCC effect removed), SEQ ID NO.15 (knob), SEQ ID NO.16 (ADCC effect removed, knob), SEQ ID NO.17 (hole), or SEQ ID NO.18 (ADCC effect removed, hole).

[0041] Furthermore, the tumor or immune cell antigen-binding domain is in the form of Fab; the tumor or immune cell antigen-binding domain is selected from antigen-binding fragments of anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, or anti-EGFR antibody. The amino acid sequence of the heavy chain VH-CH1 of the anti-PD-L1 antibody is shown in SEQ ID NO. 7; the amino acid sequence of the light chain VL-CL of the anti-PD-L1 antibody is shown in SEQ ID NO. 8. The amino acid sequence of the heavy chain VH-CH1 of the anti-CTLA4 antibody is shown in SEQ ID NO. 9; the amino acid sequence of the light chain VL-CL of the anti-CTLA4 antibody is shown in SEQ ID NO. 10. The amino acid sequence of the heavy chain VH-CH1 of the anti-PD-1 antibody is shown in SEQ ID NO. 11; the amino acid sequence of the light chain VL-CL of the anti-PD-1 antibody is shown in SEQ ID NO. 12. The amino acid sequence of the heavy chain VH-CH1 of the anti-EGFR antibody is shown in SEQ ID NO. 13; the amino acid sequence of the light chain VL-CL of the anti-EGFR antibody is shown in SEQ ID NO. 14. The amino acid sequence of the immunoglobulin Fc domain in the combination of the tumor or immune cell antigen-binding domain and the immunoglobulin Fc domain is shown in SEQ ID NO. 2 or SEQ ID NO. 3.

[0042] In this invention, the linker peptide is either an insoluble linker peptide or a linker peptide that can be recognized and cleaved by matrix metalloproteinases. The amino acid sequence of the insoluble linker peptide is shown in SEQ ID NO. 4; the amino acid sequence of the linker peptide that can be recognized and cleaved by matrix metalloproteinases is shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0043] In this invention, the amino acid sequence of the homodimer from the N-terminus to the C-terminus is as shown in SEQ ID NO. 19 or SEQ ID NO. 20; or, the homodimer comprises a first structural sequence and a second structural sequence, wherein the amino acid sequence of the first structural sequence from the N-terminus to the C-terminus is as shown in any one of SEQ ID NO. 21 to 26, and the amino acid sequence of the second structural sequence is as shown in SEQ ID NO. 8.

[0044] Specifically, the homodimer is: (1) Homodimer PNGase F-Fc (wild type) The homodimer consists of, from N-terminus to C-terminus, human IgG1 Fc and a linker peptide (G4S). n The peptide-N-glycosidase PNGase F fragment, with the amino acid sequence shown in SEQ ID NO. 19.

[0045] (2) Homodimer PNGase F-Fc (removal of ADCC effect) The homodimer, from N-terminus to C-terminus, consists of: human IgG1 Fc fragment and linker peptide (G4S). n PNGase F, a peptide-N-glycosidase, has the amino acid sequence shown in SEQ ID NO. 20.

[0046] (3) Homodimer antiPDL1-Fc (wild-type)-PNGase F The first structural sequence of the homodimer, from N-terminus to C-terminus, consists of: an antiPDL1 VH-CH1 fragment, a human IgG1 Fc fragment, and a linker peptide (G4S). n The peptide-N-glycosidase PNGase F has the amino acid sequence shown in SEQ ID NO. 21, and the second structural sequence is the antiPDL1 VL-CL fragment, the amino acid sequence of which is shown in SEQ ID NO. 8.

[0047] (4) Homodimer antiPDL1-Fc (removes ADCC effect)-PNGase F The first structural sequence of the homodimer, from N-terminus to C-terminus, consists of: an antiPDL1 VH-CH1 fragment, a human IgG1 Fc fragment, and a linker peptide (G4S). n The peptide-N-glycosidase PNGase F has the amino acid sequence shown in SEQ ID NO. 22, and the second structural sequence is the antiPDL1 VL-CL fragment, the amino acid sequence of which is shown in SEQ ID NO. 8.

[0048] (5) Homodimer antiPDL1-Fc (wild-type)-MMP9-PNGase F The homodimer consists of, from N-terminus to C-terminus, the following sequence: antiPDL1 VH-CH1 fragment, human IgG1 Fc fragment, a sequence linker peptide that can be recognized and cleaved by matrix metalloproteinase MMP9, and peptide-N-glycosidase PNGase F, with the amino acid sequence shown in SEQ ID NO. 23. The second structural sequence is the antiPDL1 VL-CL fragment, with the amino acid sequence shown in SEQ ID NO. 8.

[0049] (6) Homodimer antiPDL1-Fc (removes ADCC effect)-MMP9-PNGase F The homodimer consists of, from N-terminus to C-terminus, the following sequence: antiPDL1 VH-CH1 fragment, human IgG1 Fc fragment, a sequence linker peptide that can be recognized and cleaved by matrix metalloproteinase MMP9, and peptide-N-glycosidase PNGase F, with the amino acid sequence shown in SEQ ID NO. 24. The second structural sequence is the antiPDL1 VL-CL fragment, with the amino acid sequence shown in SEQ ID NO. 8.

[0050] (7) Homodimer antiPDL1-Fc (wild-type)-MMP14-PNGase F The homodimer consists of, from N-terminus to C-terminus, the following sequence: antiPDL1 VH-CH1 fragment, human IgG1 Fc fragment, a sequence linker peptide that can be recognized and cleaved by matrix metalloproteinase MMP14, and peptide-N-glycosidase PNGase F, with the amino acid sequence shown in SEQ ID NO. 25. The second structural sequence is the antiPDL1 VL-CL fragment, with the amino acid sequence shown in SEQ ID NO. 8.

[0051] (8) Homodimer antiPDL1-Fc (removes ADCC effect)-MMP14-PNGase F The homodimer consists of, from N-terminus to C-terminus, the following sequence: antiPDL1 VH-CH1 fragment, human IgG1 Fc fragment, a sequence linker peptide that can be recognized and cleaved by matrix metalloproteinase MMP14, and peptide-N-glycosidase PNGase F, with the amino acid sequence shown in SEQ ID NO. 26. The second structural sequence is the antiPDL1 VL-CL fragment, with the amino acid sequence shown in SEQ ID NO. 8.

[0052] In this invention, the heterodimer comprises a first monomer, a second monomer, and a third monomer. The amino acid sequence of the first monomer is shown in any one of SEQ ID NO. 27-38, the amino acid sequence of the second monomer is shown in any one of SEQ ID NO. 39-42, and the third monomer is an antiPDL1 VL-CL fragment with the amino acid sequence shown in SEQ ID NO. 8. The first monomer and the second monomer are specifically paired through a Knob-in-Hole mutation strategy.

[0053] Specifically, the first monomer of the heterodimer is: (1) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, peptide-N-glycosidase PNGase F and a linker fragment (G4S). n Human IgG1 Fc (hole) fragment, amino acid sequence as shown in SEQ ID NO. 27.

[0054] (2) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, peptide-N-glycosidase PNGase F and a linker fragment (G4S). n Human IgG1 Fc (knob) fragment, amino acid sequence as shown in SEQ ID NO. 28.

[0055] (3) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, a peptide-N-glycosidase PNGase F, a sequence linker that can be recognized and cleaved by matrix metalloproteinase MMP9, and a human IgG1 Fc (hole) fragment, with the amino acid sequence shown in SEQ ID NO. 29.

[0056] (4) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, a peptide-N-glycosidase PNGase F, a sequence linker that can be recognized and cleaved by matrix metalloproteinase MMP9, and a human IgG1 Fc (knob) fragment, with the amino acid sequence shown in SEQ ID NO. 30.

[0057] (5) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, a peptide-N-glycosidase PNGase F, a sequence linker that can be recognized and cleaved by matrix metalloproteinase MMP14, and a human IgG1 Fc (hole) fragment, with the amino acid sequence shown in SEQ ID NO. 31.

[0058] (6) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, a peptide-N-glycosidase PNGase F, a sequence linker that can be recognized and cleaved by matrix metalloproteinase MMP14, and a human IgG1 Fc (knob) fragment, with the amino acid sequence shown in SEQ ID NO. 32.

[0059] (7) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, peptide-N-glycosidase PNGase F and a linker fragment (G4S). n Remove the ADCC effector mutant IgG Fc (hole) fragment, the amino acid sequence of which is shown in SEQ ID NO. 33.

[0060] (8) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, peptide-N-glycosidase PNGase F and a linker fragment (G4S). n Remove the ADCC-effect mutant IgG Fc (knob) fragment, the amino acid sequence of which is shown in SEQ ID NO. 34.

[0061] (9) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, a peptide-N-glycosidase PNGase F, a sequence linker that can be recognized and cleaved by matrix metalloproteinase MMP9, and an IgG Fc (hole) fragment with ADCC effect removed, with the amino acid sequence shown in SEQ ID NO. 35.

[0062] (10) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, a peptide-N-glycosidase PNGase F, a sequence linker that can be recognized and cleaved by matrix metalloproteinase MMP9, and an IgG Fc (knob) fragment with ADCC effect removed, and the amino acid sequence is shown in SEQ ID NO. 36.

[0063] (11) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, a peptide-N-glycosidase PNGase F, a sequence linker that can be recognized and cleaved by matrix metalloproteinase MMP14, and an IgG Fc (hole) fragment with ADCC effect removed, with the amino acid sequence shown in SEQ ID NO. 37.

[0064] (12) The first monomer of the heterodimer consists of, from the N-terminus to the C-terminus, a peptide-N-glycosidase PNGase F, a sequence linker that can be recognized and cleaved by matrix metalloproteinase MMP14, and an IgG Fc (knob) fragment with ADCC effect removed, and the amino acid sequence is shown in SEQ ID NO. 38.

[0065] The second monomer of the heterodimer is: (1) The second monomer of the heterodimer consists of an antiPDL1 VH-CH1 fragment and a human IgG1 Fc (hole) fragment from the N-terminus to the C-terminus, and its amino acid sequence is shown in SEQ ID NO. 39.

[0066] (2) The second monomer of the heterodimer consists of an antiPDL1 VH-CH1 fragment and a human IgG1 Fc (knob) fragment from the N-terminus to the C-terminus, and its amino acid sequence is shown in SEQ ID NO. 40.

[0067] (3) The second monomer of the heterodimer consists of an antiPDL1 VH-CH1 fragment and an IgG Fc (hole) fragment with ADCC effect removed from the N-terminus to the C-terminus, with the amino acid sequence shown in SEQ ID NO. 41.

[0068] (4) The second monomer of the heterodimer consists of an antiPDL1 VH-CH1 fragment and an IgG Fc (knob) fragment with ADCC effect removed from the N-terminus to the C-terminus, with the amino acid sequence shown in SEQ ID NO. 42.

[0069] Both the homodimers and heterodimers mentioned above have a signal peptide attached to their N-terminus to promote secretory expression.

[0070] The aforementioned fusion protein achieves a synergistic function of targeted binding and local deglycosylation of PD-L1 by molecularly coupling a monoclonal antibody that specifically recognizes PD-L1 with the PNGase F enzyme molecule, thereby interfering with its immunosuppressive activity and enhancing the anti-tumor immune response.

[0071] Nucleic acid molecules, vectors, and host cells encoding the fusion protein of the present invention The present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the aforementioned fusion protein.

[0072] In some aspects, the present invention relates to vectors comprising nucleic acid sequences encoding as disclosed herein. In this invention, the vector can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, bacteriophage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and bacteriophage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the vector is suitable for expressing a fusion protein as disclosed herein in bacterial cells. Examples of such vectors include expression vectors such as BlueScript, pIN vectors, pET vectors, pEE (pEE12.4, pEE6.4) vectors, etc.

[0073] This invention also relates to host cells comprising the vector described herein. Therefore, this invention also relates to recombinant eukaryotic or prokaryotic host cells, such as transfected tumors, that produce the fusion protein of this invention. The fusion protein can be expressed in recombinant eukaryotic or prokaryotic host cells, such as transfected tumors, producing the fusion protein as defined herein.

[0074] Examples of host cells include yeast, bacteria, plant and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NS0 cells or lymphocytes.

[0075] Preparation methods of fusion proteins This invention provides a method for preparing the above-mentioned fusion protein, comprising the following steps: Construct an expression vector containing the gene encoding the fusion protein; Host cells containing the expression vector are constructed by transient or stable transfection of host cells; The fusion protein was isolated and purified from the host cell.

[0076] The fusion protein can be isolated and purified by collecting the supernatant and purifying it by protein A / G affinity chromatography and molecular sieve chromatography.

[0077] Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising the above-described fusion protein and a pharmaceutically acceptable carrier.

[0078] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the fusion protein disclosed herein enhances the immune response to the antigen. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, combinations or more of various components known in the art.

[0079] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl arsenate.

[0080] Pharmaceutically acceptable carriers may include, for example, aqueous carriers such as sodium chloride injection, Ringer's solution, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's solution; non-aqueous carriers such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents or antifungal concentrations of antimicrobial agents; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); blocking or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethylene glycol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions containing phenol or cresol, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzyl chloride in multi-dose containers. Suitable excipients may include, for example, water, saline, dextran, glycerol or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitol monolaurate, triethanolamine oleate or cyclodextrin.

[0081] Application of the present invention This invention provides the use of the above-described fusion protein or the above-described pharmaceutical composition in the preparation of antitumor drugs, wherein the use includes one or more of the following: (i) Preparation of antitumor drugs; (ii) Preparation of antitumor drugs for use in combination with immune checkpoint inhibitors; (iii) Preparation of antitumor drugs for use in combination with T cell adoptive transfer; (iv) To prepare drugs for postoperative adjuvant immunotherapy to prevent tumor recurrence or metastasis after surgery; (v) To prepare combination therapy drugs to enhance the effects of radiotherapy or chemotherapy; (vi) Prepare pharmaceutical compositions for the treatment of patients with drug-resistant or recurrent tumors.

[0082] A therapeutically effective amount of the fusion protein disclosed herein can be administered to a patient (e.g., a human) who requires treatment.

[0083] The tumors described in this invention can be malignant or benign, and can be primary or secondary. These tumors can be solid tumors or hematologic malignancies. Solid tumors include, but are not limited to: melanoma of the skin, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma (such as clear cell subtype), hepatocellular carcinoma, colon cancer, rectal cancer, breast cancer, prostate cancer, pancreatic cancer, bladder cancer, thyroid cancer, endometrial cancer, esophageal cancer, gastric cancer, bile duct cancer, ovarian cancer, etc.; hematologic malignancies include leukemia, lymphoma, multiple myeloma, etc.

[0084] The fusion protein of the present invention or the above-described pharmaceutical composition can also be used in combination with other drugs or methods, such as immune checkpoint inhibitors, T-cell adoptive transfer, etc. Preferably, the adoptive transferred T cells are anti-tumor specific T cells; more preferably, the T cells are anti-tumor CAR-T, TCR-T or other structurally similar cells, or anti-tumor stem cells.

[0085] The technical solution of the present invention will be further described below with reference to specific embodiments. These embodiments are provided by way of example and are not intended to limit the present invention.

[0086] The experimental materials and methods used in the following examples are summarized below: 1. Bacterial strains and plasmids Bacterial strains: Top10 E. coli, DH5α E. coli competent cells (Beijing TransGen Biotechnology Co., Ltd.) Plasmid: pEE12.4-IgGκ, containing the signal peptide of mouse IgGκ, for the expression of fusion protein.

[0087] All primers used in the experiment were designed using DNAMAN software and synthesized by Genewiz.

[0088] 2. Laboratory animals Wild-type C57BL / 6 and BALB / c-nude mice were purchased from Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. Unless otherwise specified, all mice used in the experiments were 8-10 weeks old females.

[0089] All mice were housed in a specific pathogen-free (SPF) barrier environment. Animal husbandry and experimental procedures followed the relevant regulations of the Animal Management Committee of China Pharmaceutical University.

[0090] 3. Cell lines MC38 is a C57BL / 6 background mouse colorectal cancer cell line, and B16 is a C57BL / 6 background mouse melanoma cell line. All these cell lines were cultured in DMEM complete medium (containing 10% inactivated fetal bovine serum, 2 mmol / L L-glutamine, 0.1 mmol / L non-essential amino acids, 100 U penicillin, and 100 μg / ml streptomycin).

[0091] The TIB-210™ hybridoma cell line (ATCCTIB-210) is used to express CD8. + T-cell deletion antibody (clone: ​​2.43).

[0092] The TIB-207™ hybridoma cell line (ATCC-TIB-207) is used to express CD4. + T-cell deletion antibody (clone: ​​GK1.5).

[0093] The HB-197™ hybridoma cell line (ATCC-HB-197) is used to express an antibody that blocks FcγRII / III in mice (clone: ​​2.4G2).

[0094] The FreeStyle™ 293F cell line (Invitrogen) is a suspension cell line derived from the HEK293 cell line. It is cultured in SMM293-TII or CD OptiCHO™ medium and is mainly used for transient transfection expression of fusion proteins.

[0095] The cell lines were cultured in RPMI 1640 complete medium (containing 10% inactivated fetal bovine serum, 2 mmol / L L-glutamine, 0.1 mmol / L non-essential amino acids, 100 U penicillin and 100 μg / ml streptomycin, 100 IU / ml recombinant IL-2).

[0096] 4. Mouse tumor inoculation and treatment (1) Tumor inoculation and measurement: Establishment of tumor model: 5×10 5 One MC38 single cell was suspended in 100 μL PBS and subcutaneously injected into the back of C57BL / 6 mice; When performing a re-challenge experiment on mice with regressed tumors using the same type of tumor cells, the number of tumor cells inoculated was 5 times that at the initial tumor modeling site, and the inoculation site was subcutaneous on the opposite side of the mouse's back. Tumor size was monitored twice a week, and the long diameter (a), short diameter (b), and height (c) of the tumor were measured using calipers. The mouse tumor volume was calculated as a × b × c / 2.

[0097] (2) Treatment: Antibodies or antibody fusion proteins are administered via intraperitoneal injection, and some experiments also use intratumoral administration. Specific dosages will be described in the specific experiments.

[0098] 5. Monoclonal antibody preparation (mouse ascites method): The CD4+ T cell deletion antibodies GK1.5 and CD8 used in the experiment + The T-cell deletion antibody TIB210 and the FcRII / III blocking antibody are derived from the corresponding hybridoma cells (TIB-210TM, TIB-207TM, HB-197TM) and were produced and purified in our laboratory.

[0099] 6. Cell deletion in mice CD4 + T cells, CD8 + Deletion of T cells: One day prior to fusion protein treatment, 200 μg of GK1.5 or TIB210 antibody was injected intraperitoneally to remove CD4. + T cells, CD8 + T cells were injected every 3 days thereafter, with the frequency adjusted according to the treatment cycle. Deletion efficiency was assessed by flow cytometry.

[0100] 7. T cell migration blockade FTY720 (purchased from Sigma) is an immunosuppressant that reduces the migration of T cells from lymphoid organs into the peripheral bloodstream. In this invention, FTY720 blockade was performed at different stages of mouse tumor inoculation to alter the tumor microenvironment. Blockade was performed during mouse tumor treatment: 25 μg of FTY720 was injected intraperitoneally one day before tumor treatment, followed by 20 μg intraperitoneally every other day, with the blockade duration depending on the treatment cycle. This resulted in no new T cells migrating into the tumor tissue during tumor treatment. The FTY720 blockade protocol allows for the investigation of the importance of lymphocytes infiltrating tumor tissue.

[0101] Example 1 This embodiment provides the design, construction, purification, and production of glycosidase fusion proteins. This embodiment fuses the PD-L1 monoclonal antibody (αPD-L1) with the N-glycoprotein deglycosylation enzyme PNGase F, which targets and blocks the highly glycosylated PD-L1 in the tumor microenvironment and deglycosylates it, thereby promoting its degradation (e.g., Figure 1(As shown in A). A high-affinity PD-L1 monoclonal antibody sequence was used to ensure its specific binding to PD-L1. A flexible linker peptide (G4S)3 was introduced between the Fc fragment of αPD-L1 and PNGase F, and PNGase F enzyme with highly efficient deglycosylation activity was selected to ensure that its enzyme activity was not affected after fusion, thus maintaining the independent function of both. A signal peptide was added at the N-terminus to promote secretory expression.

[0102] The fusion protein constructed in this embodiment is in the form of a homodimer, which includes a first structural sequence and a second structural sequence. The first structural sequence, from the N-terminus to the C-terminus, consists of: an antiPDL1 VH-CH1 fragment, a human IgG1 Fc fragment, and a linker peptide (G4S). n The peptide-N-glycosidase PNGase F has the amino acid sequence shown in SEQ ID NO. 21, and the second structural sequence is the antiPDL1 VL-CL fragment, the amino acid sequence of which is shown in SEQ ID NO. 8.

[0103] The fusion protein gene was constructed into a eukaryotic expression vector and transiently transfected into 293F cells. Seven days after transfection, the cell supernatant was collected and purified using protein A. The purified protein was quantified by ELISA and Nanodrop. Purity was determined by SDS-PAGE (2 μg per sample).

[0104] The specific steps are as follows: a. Transient transfection for rapid expression of the target protein (1) Cell resuscitation: Freestyle 293F cells were revived at a rate of 3 × 10⁻⁶ cells / year. 7 Cells were cryopreserved at a concentration of [number] cells / ml (cryopreservation medium: SMM 293-TII medium containing 10% DMSO). After removal from liquid nitrogen, the cells were rapidly thawed in a 37°C water bath and added to centrifuge tubes containing 10 ml of SMM 293-TII medium. The cells were centrifuged at 100 g for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 30 ml of SMM 293-TII medium and cultured at 37°C, 8% CO2, 135 rpm. Large-scale culture was then carried out over several days.

[0105] (2) Two days before transfection, prepare 293F cells in suspension culture for transient transfection (200 ml), with a seeding density of 0.6-0.8 × 10⁻⁶. 6 cells / ml.

[0106] (3) Two days later, count the cells in the suspension to be transfected. The expected cell density is 4 × 10⁻⁶. 6 The cells were counted at 1000 rpm for 5 minutes, and the supernatant was discarded.

[0107] (4) Resuspend the cells in 50 ml of fresh Freestyle293 media, centrifuge at 100 g for 5 min, and discard the supernatant.

[0108] (5) Resuspend 293F cells in 200ml of Freestyle 293 media.

[0109] (6) Dilute 200 μg of plasmid with 4 ml of Freestyle 293 media medium and filter it with a 0.22 μM filter to remove bacteria.

[0110] (7) Dilute 800 μg PEI (stock solution 2 mg / ml) with 4 ml Freestyle 293 medium and filter sterilize using a 0.22 μM filter. Immediately afterward, mix 4 ml of plasmid and 4 ml of PEI and let stand at room temperature for 5 minutes.

[0111] (8) Add the plasmid / PEI mixture dropwise to the cell suspension and incubate at 37°C, 8% CO2, and 85 rpm.

[0112] (9) After 4 h, add 200 ml of EX-CELL™ 293 medium and 2 mM Glutamine, and add 50 ug / L LONG™ R3IGF-1 growth factor at the same time. Adjust the rotation speed to 135 rpm and continue culturing.

[0113] (10) Add 3.8 mM VPA, a cell proliferation inhibitor, 24 hours later. Collect the supernatant 6-8 days after transfection (when cell survival rate is less than 70%) for the next experiment.

[0114] b. Purification of the target protein using Protein A (1) Sample preparation: Transfer the suspended cell culture medium to a 500 ml centrifuge bucket and centrifuge at 8,000 rpm for 40 minutes to discard the precipitate. Filter the supernatant through a 0.45 μm filter to remove impurities, and then add NaN3 to a final concentration of 0.05% to prevent bacterial contamination during the purification process.

[0115] (2) Assemble the chromatography column: Take an appropriate amount of Protein A Agarose (calculated as 20 mg human Fc fusion protein per 1 ml Protein A purification) and add it to the chromatography column. Let it stand at room temperature for about 10 min. After Protein A and 20% ethanol solution separate into layers, open the bottom outlet and let the ethanol solution flow out slowly by gravity.

[0116] (3) Rinse and equilibrate the chromatography column with 10 column volumes of distilled water and binding buffer, respectively.

[0117] (4) Load the sample using a constant flow pump at a flow rate of 10 times the column volume / hour, collect the flow-through liquid, and repeat the loading twice.

[0118] (5) Rinse the column with more than 10 column volumes of binding buffer to remove impurities and rinse until no protein is detected in the effluent.

[0119] (6) Elution was performed using elution buffer. The eluent was collected in separate tubes, one tube for every 1 ml of eluent. The elution peak was observed using a protein indicator solution (Bio-Rad protein assay). The collected tubes of the elution peak were mixed and neutralized with an appropriate amount of neutralization buffer.

[0120] (7) Use a concentrated centrifuge tube to replace the target protein solution with the required buffer, and determine the protein concentration by SDS-PAGE electrophoresis and NanoDrop2000.

[0121] (8) After elution, rinse the column with 20 column volumes of distilled water, then rinse the column with 10 column volumes of 20% ethanol. Finally, the ethanol solution should be used to immerse the gel medium and stored at 4°C.

[0122] The SDS-PAGE electrophoresis results of αPD-L1-PNGase F protein showed (e.g.) Figure 1 As shown in B), this fusion protein has high purity and can correctly express the light chain of the AntiPDL1 antibody and the heavy chain portion of the fusion protein.

[0123] Example 2 This embodiment provides intratumoral and systemic injection of the αPD-L1-PNGase F fusion protein from Example 1 to verify the antitumor effect of αPD-L1-PNGase F.

[0124] First, C57BL6 mice were subcutaneously inoculated with 5 × 10⁵ cells / mL. 5 MC38 tumor cells were used in tumor-bearing mice, and treatment began on day 12 (n=5). Mice were treated with 50 μg of the fusion protein via intratumoral injection every 3 days for 3 consecutive treatments. Tumor volume was measured (volume = length × width × height / 2). αPD-L1 and PNGase F-Fc were used as controls. Results are as follows. Figure 2As shown, intratumoral administration of the αPD-L1-PNGase F fusion protein exhibited good antitumor effects. Since both intratumoral injection of PNGase F and αPD-L1-PNGase F showed significant tumor growth inhibition, to better compare the antitumor activities of PNGase F-Fc and αPD-L1-PNGase F, we treated mice in the MC38 subcutaneous tumor model via systemic administration. C57BL6 mice were subcutaneously inoculated with 5 × 10⁶ phosphate phosphate molecules. 5 MC38 tumor cells were administered via intraperitoneal injection on day 11 post-inoculation, every 3 days for a total of 3 treatments, after which tumor volume was measured. Figure 3 The results showed that, when administered intraperitoneally at equimolar doses of PNGase F and αPD-L1-PNGase F, αPD-L1-PNGase F exhibited significantly better antitumor efficacy than PNGase F, highlighting the importance of αPD-L1 targeting. In conclusion, our designed αPD-L1-PNGase F fusion protein demonstrated superior antitumor efficacy when administered systemically.

[0125] Example 3 This embodiment provides the relationship between the antitumor activity of the αPD-L1-PNGase F fusion protein and the dosage in Example 1.

[0126] To determine whether the antitumor effect of αPD-L1-PNGase F is dose-dependent, C57BL / 6 mice were subcutaneously injected with 5 × 10⁶ αPD-L1-PNGase F. 5 MC38 tumor cells were injected intraperitoneally with 25, 50, and 75 μg of the fusion protein on day 13 after tumor inoculation, with PBS as a control.

[0127] like Figure 4 The results showed that the fusion protein treatment was most effective at a dose of 75 μg. Furthermore, the antitumor activity of αPD-L1-PNGase F exhibited a dose-dependent relationship within the dose range of 0–75 μg.

[0128] Meanwhile, to investigate the effect of the fusion protein on mouse body weight, C57BL / 6 mice were subcutaneously inoculated with 5 × 10⁶ mol / L protein. 5 MC38 tumor cells were intraperitoneally injected with 6, 17, 50, 150, and 450 μg of αPD-L1-PNGaseF fusion protein on day 13 post-inoculation. Figure 5 As shown, the results indicate that the fusion protein exhibits good safety, with weight loss only occurring in mice at a dose of 450 μg.

[0129] Example 4 This embodiment provides a comparison of the antitumor efficacy of the αPD-L1-PNGase F fusion protein of Example 1 with PD-L1 monoclonal antibody, PNGase F, PD-L1 monoclonal antibody and PNGase F in combination (αPD-L1+PNGase F) and enzyme activity mutant αPD-L1-PNGase F (αPD-L1-PNGase F-mut).

[0130] C57BL / 6 mice were subcutaneously inoculated with 5×10 5 MC38 tumor cells, until the tumor volume reaches 150 mm 3 At approximately 3:00 AM, patients were given 75 μg of αPD-L1-PNGase F and equimolar doses of αPD-L1 monoclonal antibody, PNGase F, αPD-L1+PNGase F and αPD-L1-PNGase F-mut intraperitoneally every 3 days for 3 consecutive treatments. The control group received PBS with the same administration method and frequency.

[0131] Figure 6 The results showed that the therapeutic effect of the αPD-L1-PNGase F fusion protein was superior to that of PD-L1 monoclonal antibody, PNGase F, and the combination of αPD-L1 and PNGase F, indicating that αPD-L1 and PNGase F in the fusion protein have a synergistic effect. Furthermore, mutations in the PNGase F enzyme activity in αPD-L1-PNGase F can completely eliminate the anti-tumor effect of αPD-L1-PNGase F, indicating that the therapeutic effect of the fusion protein depends on the enzyme activity of PNGase F.

[0132] Subcutaneous inoculation with 5 × 10 NSG mice (highly immunodeficient tool mice) 5 MC38 tumor cells were intratumorally injected with 50 μg of PNGase F-Fc fusion protein on days 8, 11, and 14. Figure 7 As shown, after treatment, the PNGase F-Fc fusion protein did not exhibit anti-tumor activity in NSG mice, indicating that the PNGase F enzyme had no therapeutic effect in immunodeficient mice.

[0133] Example 5 The efficacy of αPD-L1-PNGase F is independent of tumor-specific PDL1 expression.

[0134] To investigate whether the antitumor effect of the αPD-L1-PNGase F fusion protein in Example 1 depends on PD-L1 expression in tumor cells, we constructed the MC38-PD-L1-KO tumor cell line and subcutaneously seeded 5 × 10⁶ cells / year. 5 MC38-PD-L1-KO tumor cells, until the tumor volume grows to 100 mm.3 At approximately 10:00 AM, patients were treated with intraperitoneal injections of equimolar doses of αPD-L1 and αPD-L1-PNGase F fusion protein, respectively, every three days. After three administrations, tumor growth curves were monitored to evaluate and compare the antitumor efficacy of the three groups. Figure 8 The results showed that the αPD-L1-PNGase F fusion protein still had anti-tumor efficacy after PD-L1 knockout in tumor cells, indicating that the anti-tumor efficacy of the αPD-L1-PNGase F fusion protein did not depend on the PD-L1 expression level of tumor cells, suggesting that the anti-tumor efficacy of the fusion protein was more dependent on the PD-L1 expressed by immune cells in the tumor microenvironment.

[0135] Example 6 The therapeutic effect of the αPD-L1-PNGase F fusion protein depends on CD8 T cells.

[0136] To investigate which group of immune cells mediated the antitumor function of the αPD-L1-PNGase F fusion protein from Example 1, we subcutaneously injected 5 × 10⁵ cells into the back of C56BL / 6 mice. 5 MC38 tumor cells were treated 12 days post-inoculation with an intraperitoneal injection of 75 μg of αPD-L1-PNGase F fusion protein. One day prior to treatment, 200 μg of CD4 and CD8T deletion antibodies (clone numbers: GK1.5, TIB210, prepared in our laboratory) were injected intraperitoneally. Deletion efficiency was assessed, and the results showed that the antibodies effectively cleared CD4 and CD8T from peripheral blood. + T cells. CD8 deletion occurs while using fusion protein therapy. + After T-cell administration, the anti-tumor effect completely disappeared, indicating that the drug's action depends on CD8. + T cells (such as T cells) Figure 9 (As shown).

[0137] Example 7 The therapeutic effect of αPD-L1-PNGase F fusion protein depends on intratumoral infiltration of CD8. + T cells.

[0138] To further investigate the CD8 inhibitors relied upon by the αPD-L1-PNGase F fusion protein of Example 1 + Do T cells function because they are T cells residing in the tumor itself, or because drugs activate T cells outside the tumor that then migrate into the tumor to exert their effects? We used FTY720, an inhibitor that suppresses T cell migration from lymph nodes to the periphery, to block the migration of T cells from lymph nodes to the tumor, thereby maintaining the stability of T cells within the tumor.

[0139] C57BL / 6 mice were subcutaneously injected with 5 × 10⁵ mice on their backs.5 MC38 tumor cells were intraperitoneally injected with 75 μg of the fusion protein on days 12, 15, and 18 post-tumor inoculation. In the FTY720 treatment group, 25 μg of FTY720 was intraperitoneally injected on day 11 post-tumor inoculation, followed by injections of 20 μg of FTY720 on days 13, 15, 17, 19, and 21.

[0140] like Figure 10 As shown, even after FTY720 blocked T cell migration, the fusion protein still exhibited a strong anti-tumor effect. These results indicate that the anti-tumor effect of the fusion protein depends on tumor-resident T cells, rather than peripherally migrated T cells.

[0141] Example 8 The therapeutic effect of the αPD-L1-PNGase F fusion protein depends on the secretion of IFN-γ.

[0142] To further investigate the antitumor mechanism of the αPD-L1-PNGase F fusion protein from Example 1, we subcutaneously injected 5 × 10⁵ mmol / L into the back of C57BL / 6 mice. 5 MC38 tumor cells were intraperitoneally injected with 75 μg of the fusion protein on days 12, 15, and 18 post-inoculation. An αIFN-γ deletion antibody was administered the day before treatment. Figure 11 The results showed that the therapeutic effect of the fusion protein was completely eliminated after the deletion of IFN-γ. These results indicate that the antitumor effect of the fusion protein depends on the action of IFN-γ.

[0143] Example 9 The therapeutic effect of αPD-L1-PNGase F treatment, which induces tumor-specific CD8+ T cells to increase αPD-L1-PNGase F, depends on the presence of CD8 T cells within the tumor.

[0144] To further verify whether the fusion protein affects intratumoral immune cell infiltration, we subcutaneously injected 5 × 10⁵ cells into the back of C57BL / 6 mice. 5 MC38-OVA tumor cells, tumor volume grew to 100 mm 3 Subsequently, 75 μg of αPD-L1-PNGase F fusion protein was administered intraperitoneally every 3 days. Eighteen hours after the second administration, 250 μg of the Brefeldin A (BFA) protein transport inhibitor was injected intraperitoneally, and tumor tissue was harvested 6 hours after BFA injection. Control group mice were given PBS. Antigen-specific CTLs were detected using tetramer staining.

[0145] like Figure 12 As shown, αPD-L1-PNGase F fusion protein treatment not only increases intratumoral CD8 levels+ The proportion and number of T cells (AB), and the induction of tumor-specific CD8 within the tumor. + Increased T cells (CD), significantly altered the CD8 / Treg ratio (E), and induced more IFNγ. + CD8T cells produce (F).

[0146] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A fusion protein with peptide-N-glycosidase as the active ingredient, characterized in that, include: (a) Peptide-N-glycosidase or its catalytically active fragment; (b) Immunoglobulin Fc domain, or a combination of tumor or immune cell antigen-binding domain and immunoglobulin Fc domain; (c) Linking peptides; The tumor or immune cell antigen-binding domain is in the form of Fab, scFv, or VHH; the peptide-N-glycosidase or its catalytically active fragment is linked to the immunoglobulin Fc domain via the linker peptide; the immunoglobulin Fc domain mediates the formation of homodimers or heterodimers of the fusion protein.

2. The fusion protein as described in claim 1, characterized in that, The peptide-N-glycosidase is selected from one or more of PNGase F, PNGase F-II, PNGase H+, PNGase Rc, PNGase A, Endo H, Endo F3, Endo S, α-fucosidase, or NGLY1; The tumor or immune cell antigen-binding domain specifically binds to one or more of the following targets: PD-L1, CTLA-4, PD-1, TIGIT, TIM3, LAG3, 41BB, ICOS, GITR, 2B4, B7H3, B7H4, EGFR, CLDN18.2, HER2, Trop2, or Nectin-4. The immunoglobulin Fc region is the natural immunoglobulin Fc or the immunoglobulin Fc that has had its ADCC effect removed through gene mutation, and its amino acid sequence is shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.16 or SEQ ID NO.18; The linker peptide is either an uncleavable linker peptide or a linker peptide that can be recognized and cleaved by matrix metalloproteinases. The amino acid sequence of the homodimer from the N-terminus to the C-terminus is shown in SEQ ID NO. 19 or SEQ ID NO. 20; or, the homodimer comprises a first structural sequence and a second structural sequence, wherein the amino acid sequence of the first structural sequence from the N-terminus to the C-terminus is shown in any one of SEQ ID NO. 21 to 26, and the amino acid sequence of the second structural sequence is shown in SEQ ID NO. 8; The heterodimer comprises a first monomer, a second monomer, and a third monomer, wherein the amino acid sequence of the first monomer is shown in any one of SEQ ID NO. 27-38, the amino acid sequence of the second monomer is shown in any one of SEQ ID NO. 39-42, and the amino acid sequence of the third monomer is shown in SEQ ID NO. 8; wherein the first monomer and the second monomer are specifically paired through a Knob-in-Hole mutation strategy.

3. The fusion protein as described in claim 2, characterized in that, The amino acid sequence of the peptide-N-glycosidase is as shown in SEQ ID NO. 1, or a variant having at least 80% of the same amino acid sequence as SEQ ID NO. 1 and retaining peptide-N-glycosidase activity; The tumor or immune cell antigen-binding domain is in the form of Fab; the tumor or immune cell antigen-binding domain is selected from antigen-binding fragments of anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, or anti-EGFR antibody. The amino acid sequence of the immunoglobulin Fc domain in the combination of the tumor or immune cell antigen-binding domain and the immunoglobulin Fc domain is shown in SEQ ID NO. 2 or SEQ ID NO. 3; The amino acid sequence of the non-cleavable linker peptide is shown in SEQ ID NO. 4; the amino acid sequence of the linker peptide that can be recognized and cleaved by matrix metalloproteinases is shown in SEQ ID NO. 5 or SEQ ID NO.

6.

4. The fusion protein as described in claim 1, characterized in that, The fusion protein, from N-terminus to C-terminus, consists of: an antiPDL1 VH-CH1 fragment, a human IgG1 Fc fragment, and a linker peptide (G4S). n The peptide-N-glycosidase PNGase F has the amino acid sequence shown in SEQ ID NO. 21, and the second structural sequence is the antiPDL1 VL-CL fragment, the amino acid sequence of which is shown in SEQ ID NO.

8.

5. An isolated nucleic acid molecule, characterized in that, It contains a nucleic acid sequence encoding the fusion protein according to any one of claims 1 to 4.

6. A carrier comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. The method for preparing the fusion protein according to any one of claims 1 to 4, characterized in that, The steps include: Construct an expression vector containing the gene encoding the fusion protein; Host cells containing the expression vector are constructed by transient or stable transfection of host cells; The fusion protein was isolated and purified from the host cell.

9. A pharmaceutical composition, characterized in that, It comprises the fusion protein as described in any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

10. The use of the fusion protein according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 9, characterized in that, The applications include one or more of the following: (i) Preparation of antitumor drugs; (ii) Preparation of antitumor drugs for use in combination with immune checkpoint inhibitors; (iii) Preparation of antitumor drugs for use in combination with T cell adoptive transfer; (iv) To prepare drugs for postoperative adjuvant immunotherapy to prevent tumor recurrence or metastasis after surgery; (v) To prepare combination therapy drugs to enhance the effects of radiotherapy or chemotherapy; (vi) Prepare pharmaceutical compositions for the treatment of patients with drug-resistant or recurrent tumors.

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