Use of selective vegfr3 inhibitors in combination with anti-pd-l1 antibodies in the treatment of lung cancer

Abstract

The present application relates to the field of tumor treatment, and particularly relates to a combination of a selective VEGFR3 inhibitor and an anti-PD-L1 antibody and application thereof in lung cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment, specifically to the combination of selective VEGFR3 inhibitors and anti-PD-L1 antibodies and their application in the treatment of lung cancer. Background Technology

[0002] Lung cancer is considered the leading cause of cancer-related death worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent pathological type. The asymptomatic nature of NSCLC often leads to late-stage diagnosis, significantly limiting treatment options. Despite advances in comprehensive treatments such as surgery, radiotherapy, and chemotherapy, the overall survival (OS) for NSCLC patients remains dishearteningly low. Therefore, identifying novel therapeutic targets and understanding their molecular mechanisms is crucial for prolonging overall survival in NSCLC patients.

[0003] The lymphatic system, composed of lymphatic vessels and lymph nodes, is essential for normal bodily physiology and also participates in the pathogenesis of various diseases, especially cancer. In recent years, the lymphatic system has been shown to play an active role in the dissemination and metastasis of primary tumors, and lymphatic vessels have become a core participant in cancer progression. Lymphatic endothelial cells are considered direct regulators of tumor immunity and immunotherapy responsiveness, but their reported roles in different cancers remain controversial. Previous studies have shown that lymphangiogenesis is a crucial step in cancer metastasis, with the VEGFC / VEGFR3 signaling pathway playing a key role. This pathway is also associated with various diseases, such as heart failure and tumor metastasis. Therefore, inhibiting the VEGFC / VEGFR3 axis has become an important strategy in cancer treatment. However, current inhibitors of lymphatic vessels in cancer are all highly selective and multi-target inhibitors. These drugs, in addition to inhibiting VEGFR3, may also inhibit various kinases, and the "non-targeted" effect may exacerbate adverse reactions. Immune checkpoint blockade (ICB) therapy is a mature immunotherapy that can significantly reduce tumors and achieve long-term disease control in various cancers. However, only a portion of patients benefit from ICB. Therefore, exploring innovative intervention strategies to control lung cancer progression has significant scientific and clinical value. Summary of the Invention

[0004] The inventors of this application unexpectedly discovered that by inhibiting lymphangiogenesis and VEGFC expression, the formation of tertiary lymphoid structures can be promoted to inhibit the progression of NSCLC, thereby providing a new combination therapy target for inhibiting lung cancer progression, and thus providing the following aspects.

[0005] In one aspect, the present invention provides the use of selective VEGFR3 inhibitors and antiPD-L1 antibodies or antigen-binding fragments thereof in the treatment of lung cancer.

[0006] In some implementations, the lung cancer is non-small cell lung cancer.

[0007] In some embodiments, the selective VEGFR3 inhibitor is T72924. T72924 has CAS: 2756668-73-0 and has the following structure:

[0008] .

[0009] In some embodiments, the anti-PD-L1 antibody is selected from B7-H1, atezolizumab, durvalumab, avelumab, envafolimab, or sugemalimab.

[0010] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, or diabody.

[0011] In some embodiments, the selective VEGFR3 inhibitor and the anti-PD-L1 antibody or its antigen-binding fragment are in separate dosage forms. A first composition is formed from the selective VEGFR3 inhibitor and a pharmaceutically acceptable carrier and / or excipient. A second composition is formed from the anti-PD-L1 antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the first and second compositions each contain an effective amount of the selective VEGFR3 inhibitor and an effective amount of the anti-PD-L1 antibody or its antigen-binding fragment, respectively.

[0012] In some embodiments, the selective VEGFR3 inhibitor and the anti-PD-L1 antibody or its antigen-binding fragment are in the same dosage form. The selective VEGFR3 inhibitor and the anti-PD-L1 antibody or its antigen-binding fragment, along with a pharmaceutically acceptable carrier and / or excipient, form a single composition. In some embodiments, the single composition comprises an effective amount of the selective VEGFR3 inhibitor and an effective amount of the anti-PD-L1 antibody or its antigen-binding fragment.

[0013] In another aspect, the present invention provides a pharmaceutical combination comprising a selective VEGFR3 inhibitor and an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0014] In some embodiments, the selective VEGFR3 inhibitor is T72924.

[0015] In some embodiments, the anti-PD-L1 antibody is selected from B7-H1, atezolizumab, durvalumab, avelumab, envafolimab, or sugemalimab.

[0016] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, or diabody.

[0017] In some embodiments, the selective VEGFR3 inhibitor and the anti-PD-L1 antibody or its antigen-binding fragment are in separate dosage forms. A first composition is formed from the selective VEGFR3 inhibitor and a pharmaceutically acceptable carrier and / or excipient. A second composition is formed from the anti-PD-L1 antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the first and second compositions each contain an effective amount of the selective VEGFR3 inhibitor and an effective amount of the anti-PD-L1 antibody or its antigen-binding fragment, respectively.

[0018] In some embodiments, the selective VEGFR3 inhibitor and the anti-PD-L1 antibody or its antigen-binding fragment are in the same dosage form. The selective VEGFR3 inhibitor and the anti-PD-L1 antibody or its antigen-binding fragment, along with a pharmaceutically acceptable carrier and / or excipient, form a single composition. In some embodiments, the single composition comprises an effective amount of the selective VEGFR3 inhibitor and an effective amount of the anti-PD-L1 antibody or its antigen-binding fragment.

[0019] The selective VEGFR3 inhibitors and anti-PD-L1 antibodies or their antigen-binding fragments described in this invention, and combinations thereof, can be formulated into dosage forms compatible with their intended routes of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation), transdermal administration (i.e., local administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or glucose. pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0020] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (wherein which water is soluble) or dispersions and sterile powders for readily preparing sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, polyoxyethylene castor oil ELTM, or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid sufficient for easy injection. It must be stable under manufacturing and storage conditions and must be preserved against contamination by microorganisms such as bacteria and fungi. Carriers can be solvents or dispersion media including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Protection against microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride, will be preferably included in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0021] The selective VEGFR3 inhibitor and anti-PD-L1 antibody or their antigen-binding fragments, or combinations thereof, described in this invention can be administered to a subject (e.g., a human) by any suitable method known in the art. For many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the selective VEGFR3 inhibitor and anti-PD-L1 antibody or their antigen-binding fragments, or combinations thereof, described in this invention are administered by intravenous injection or bolus.

[0022] Terminology Definition

[0023] In this application, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0024] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0025] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0026] As used herein, the term "selective VEGFR3 inhibitor," also known as "specific VEGFR3 inhibitor," refers to a class of biomolecules or small molecules that specifically target vascular endothelial growth factor receptor 3 (VEGFR3) and block downstream signaling pathways by inhibiting its kinase activity. Its core characteristic is selective inhibition of VEGFR3, meaning that its inhibitory efficacy against VEGFR3 is significantly higher than its efficacy against other homologous or related kinases (such as VEGFR1, VEGFR2, or other tyrosine kinases). In some embodiments, the inhibitory activity (e.g., IC50 or Ki value) of the selective VEGFR3 inhibitor against VEGFR3 is significantly lower than its inhibitory activity against other targets (such as VEGFR1 and VEGFR2), for example, a difference of ≥10-fold.

[0027] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen via at least one antigen-binding site located in its variable region. When the term "antibody" is used, unless the context explicitly indicates otherwise, it includes not only the complete antibody but also the antigen-binding fragment capable of specifically binding to a target antigen. A "complete antibody" typically consists of two pairs of polypeptide chains, each pair comprising one light chain (LC) and one heavy chain (HC). The antibody light chain can be classified as κ (kappa) and λ (lambda) light chains. The heavy chain can be classified as μ, δ, γ, α, or ε, and the isotypes of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. 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 light chain constant region consists of one domain, CL. The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding sites.

[0028] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0029] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0030] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding moiety." Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, diabody, single-domain antibody, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0031] As used herein, the term "Fd" refers to an antibody fragment consisting of VH and CH1 domains; the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region; and the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0032] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0033] As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has a variety of functions but does not participate in antigen binding. "Effective functions" mediated by the Fc domain include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phage activity; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. The Fc domain can include both native and variant Fc regions. Native Fc regions contain amino acid sequences consistent with those found in naturally occurring Fc regions, such as the native human IgG1 Fc region; the native human IgG2 Fc region; the native human IgG3 Fc region; and the native human IgG4 Fc region, as well as their naturally occurring variants. Variant Fc regions contain amino acid sequences that differ from the amino acid sequence of native Fc regions due to at least one amino acid modification. In some implementations, the variant Fc region may have altered effector functions compared to the native Fc region (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function).

[0034] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof may also be used. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0035] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc.Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0036] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as referring to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody.

[0037] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delay agents, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delay agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.

[0038] As used herein, the term "subject" includes mammals. In some embodiments, the subject refers to a human. In some embodiments, the subject (e.g., a human) has a tumor, or is at risk of having the aforementioned disease.

[0039] As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of this invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received). For the purposes of "antitumor effect," this includes, but is not limited to, reductions in tumor volume, number of cancer cells, number of metastases, increased life expectancy, reduced cancer cell proliferation, reduced cancer cell survival, or improvement in various physiological symptoms associated with cancer.

[0040] As used herein, the term "effective amount" is at least the minimum concentration required to achieve measurable improvement or prevention of a particular condition. Effective amounts as used herein can vary with factors such as the patient's disease state, age, sex, and weight. An effective amount is also the amount in which the beneficial therapeutic effect outweighs any toxic or adverse effects of the treatment. For therapeutic use, beneficial or desired outcomes include clinical results such as reducing one or more symptoms arising from the disease, improving the quality of life of those suffering from the disease, reducing the dosage of other medications needed to treat the disease, enhancing the effect of another medication (e.g., via targeted therapy), delaying disease progression, and / or prolonging survival. In the case of cancer or tumors, an effective amount of a drug may be effective in reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., to some extent slowing or desiredly stopping) the infiltration of cancer cells into peripheral organs; inhibiting (i.e., to some extent slowing and desiredly stopping) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating one or more symptoms associated with the condition to some extent. An effective amount may be administered in one or more applications. For the purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a therapeutic treatment.

[0041] Beneficial effects of the invention

[0042] This application provides an innovative intervention strategy combining a specific VEGFR3 inhibitor with a PD-L1 monoclonal antibody. By inhibiting lymphangiogenesis and VEGFC expression, it can promote the formation of tertiary lymphoid structures and thus inhibit the progression of NSCLC. This provides a new combination therapy target for inhibiting lung cancer progression and has important scientific significance and clinical value for controlling lung cancer progression.

[0043] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0044] Figures 1A-1J VEGFR3 inhibitors combined with PD-L1 monoclonal antibodies significantly inhibited tumor progression in allogeneic orthotopic xenografts. A: Schematic diagram of allogeneic orthotopic model construction and treatment. B: In vivo imaging of mouse tumor formation and treatment process. C: Growth curves of mice after treatment in different treatment groups, n=7. D: Survival curves (OS) of mice in different treatment groups after treatment, n=7. E: Immunohistochemistry of mouse lung tissue showed a decrease in the expression of lymphangiogenesis-related genes after combined treatment. FH: HE staining of mouse lungs (F) showed a significant reduction in both tumor area (H) and number (G) after combined treatment. IJ: IF staining (I) and number (J) of tertiary lymphoid structures (TLS) in mouse lung tissue after treatment in different treatment groups showed that combined treatment significantly promoted the formation of tertiary lymphoid structures. Scale bar: 50μm.

[0045] Figures 2A-2J VEGFR3 inhibitors combined with PD-L1 monoclonal antibodies significantly inhibited tumor progression in xenograft orthotopic tumors. A: Schematic diagram of xenograft orthotopic model construction and treatment. B: In vivo imaging of mouse tumor formation and treatment process. C: Growth curves of mice after treatment in different treatment groups, n=7. D: Survival curves (OS) of mice in different treatment groups after treatment, n=7. E: Immunohistochemistry of mouse lung tissue showed a decrease in the expression of lymphangiogenesis-related genes after combined treatment. FH: HE staining of mouse lungs (F) showed a significant reduction in tumor area (H) and number (G) after combined treatment. IJ: IF staining (I) and number (J) of tertiary lymphoid structures (TLS) in mouse lung tissue after treatment in different treatment groups showed that combined treatment significantly promoted the formation of tertiary lymphoid structures. Scale bar: 50μm.

[0046] Figures 3A-3C Comparison of the inhibitory efficiencies of VEGFR3 inhibitors T72924 and MAZ51 on lymphoendothelial cell proliferation and tubular formation. A: Lymphoendothelial cell proliferation curves after LLC cell supernatant stimulation and treatment with different inhibitors. B: Lymphoendothelial cell proliferation curves after A549 cell supernatant stimulation and treatment with different inhibitors. C: Tube-forming ability of lymphoendothelial cells after treatment with different inhibitors. Scale bar: 20 μm. Detailed Implementation

[0047] The invention will now be described in the following non-limiting embodiments.

[0048] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0049] Example 1: VEGFR3 inhibitors combined with PD-L1 monoclonal antibodies can significantly inhibit tumor progression in allogeneic orthotopic xenografts.

[0050] In a mouse orthotopic tumor model, the combined use of a VEGFR3 inhibitor (VEGFR-3-IN-1, T72924, TargetMol) and a PD-L1 monoclonal antibody (B7-H1, be0285, BioXCell) was validated to inhibit the progression of non-small cell lung cancer. Model construction and dosing regimen are as follows: Figure 1A As shown, 1*10 6 Lewis lung cancer cells LLC (obtained from ATCC) were injected into the lungs of C57BL / 6 mice (obtained from Guangdong Yaokang) to construct a mouse orthotopic allogeneic tumor model. Eight-week-old female nude mice were randomly divided into four groups: 1) orthotopic lung cancer model without treatment; 2) orthotopic lung cancer model + PD-L1 monoclonal antibody alone treatment group; 3) orthotopic lung cancer model + VEGFR3 inhibitor alone treatment group; 4) orthotopic lung cancer model + PD-L1 monoclonal antibody and VEGFR3 inhibitor combined treatment group. In vivo imaging monitoring was performed every 5 days after tumor formation. Figure 1B ), and plotted mouse tumor growth curves ( Figure 1C ) and survival curve ( Figure 1D We found that the combination therapy group significantly inhibited tumor growth compared to the untreated group. Simultaneously, Western blotting (WB) was used to detect lymphangiogenesis-related genes. The results showed that the expression of these genes was downregulated after combination therapy. (Antibodies used included VEGFC (60004-1-1g, Proteintech, 1:5,000), VEGFR3 (AF743, R&DSystems, 1:2000), PROX1 (ab199359, Abcam, 1:1000), PODOPLANIN (11-009, Millipore, 1:2000), MECA79 (sc19602, Santa Cruz, 1:2000), GAPDH (ab9482, Abcam, 1:5000)). Figure 1EThe collected mouse lung tissue was sectioned and stained with hematoxylin and eosin (HE). Figure 1F We found that compared to the untreated group, both treatment groups also showed a reduction in tumor area and number of lesions after treatment, but the combined treatment group showed the most significant reduction in tumor area and number of lesions after treatment. Finally, to verify the effect of combination therapy on the formation of tertiary lymphoid structures, IF technology (antibodies used included NAPSINA (ab166619, Abcam, 1:2000), CD3 (ab5690, Abcam 1:2000), B220 (103202, Biolegend, 1:200), Alexa Fluor 488 (ab150129, 150073 and ab150077 Abcam, 1:2000), Alexa Fluor 555 (ab150074 and 150130 Abcam, 1:2000), Alexa Fluor 647 (ab150077, Abcam, 1:2000)) was used to verify the formation of tertiary lymphoid structures. We found that compared with the untreated group, the combination therapy group significantly promoted the formation of tertiary lymphoid structures after treatment. Figure 1I -J). This study confirmed that the combination of VEGFR3 inhibitors and PD-L1 monoclonal antibodies exerts a tumor-suppressive effect on non-small cell lung cancer by promoting the formation of tertiary lymphoid structures, suggesting that it could serve as a new potential combination therapy target for non-small cell lung cancer.

[0051] Example 2: VEGFR3 inhibitors combined with PD-L1 monoclonal antibodies can significantly inhibit tumor progression in xenografts.

[0052] The effect of the combination of VEGFR3 inhibitor and PD-L1 monoclonal antibody on the progression of non-small cell lung cancer was verified in a mouse xenograft orthotopic tumor model. Model construction and dosing regimen are as follows: Figure 2A As shown, 2*10 6 Human lung cancer A549 cells (obtained from ATCC) were intravenously injected into C57BL / 6 mice (obtained from Guangdong Yaokang) to construct a xenogeneic orthotopic tumor model. Eight-week-old female nude mice were randomly divided into four groups: 1) orthotopic lung cancer model without treatment; 2) orthotopic lung cancer model + PD-L1 monoclonal antibody monotherapy group; 3) orthotopic lung cancer model + VEGFR3 inhibitor monotherapy group; 4) orthotopic lung cancer model + PD-L1 monoclonal antibody and VEGFR3 inhibitor combination therapy group. In vivo imaging monitoring was performed every 5 days after tumor formation. Figure 2B ), and plotted mouse tumor growth curves ( Figure 2C ) and survival curve ( Figure 2DWe found that compared to the untreated group, both single-treatment groups showed a trend of slower tumor growth, but the combination therapy group showed the most significant inhibition of tumor growth. Simultaneously, Western blotting analysis of lymphangiogenesis-related genes revealed that the expression of these genes was downregulated after combination therapy. Figure 2E The collected mouse lung tissue was sectioned and stained with hematoxylin and eosin (HE). Figure 2F We found that compared to the untreated group, the combination therapy group showed a significant reduction in tumor area and number of lesions after treatment. Finally, to verify the effect of combination therapy on the formation of tertiary lymphoid structures, we used IF technology to verify the formation of tertiary lymphoid structures. We found that compared to the untreated group, the combination therapy group significantly promoted the formation of tertiary lymphoid structures after treatment. Figure 2I -J). This study confirmed that the combination of VEGFR3 inhibitors and PD-L1 monoclonal antibodies exerts a tumor-suppressive effect on non-small cell lung cancer by promoting the formation of tertiary lymphoid structures, suggesting that it could serve as a new potential combination therapy target for non-small cell lung cancer.

[0053] Example 3: Comparison of the inhibitory efficiency of VEGFR3 inhibitor T72924 and VEGFR3 inhibitor MAZ51 on lymphoendothelial cell proliferation and tube formation.

[0054] The effects of two VEGFR3 inhibitors on lymphoendothelial cell proliferation and tubulation were validated in LLC and A549 cell models. Different cell types (5 x 10 cells per well) were seeded in 96-well plates. 2 Cells were randomly divided into three groups: 1) a control group without inhibitors, 2) treatment groups receiving different tumor cell supernatants plus the inhibitor MAZ51 (HY-116624, MCE, 2.5uM), and 3) treatment groups receiving different tumor cell supernatants plus the inhibitor T72924 (100nM). Cell proliferation was monitored every 24 hours using CCK-8, and cell proliferation curves were plotted. Figure 3A (-B) We found that compared to the control group, cell growth slowed down in both treatment groups, but the cell proliferation reduction was most significant in the group treated with the inhibitor T72924. In the tube formation assay, different cell types (2*102 cells per well) were seeded in 24-well plates. 3 The tumors were randomly divided into three groups: 1) a control group without inhibitors, 2) treatment groups with different tumor supernatants plus the inhibitor MAZ51 (HY-116624, MCE, 2.5uM), and 3) treatment groups with different tumor supernatants plus the inhibitor T72924 (100nM). Tube formation assays performed at 6 hours also showed that the addition of the inhibitor T72924 resulted in the most significant decrease in tube formation ability. Figure 3C The study confirmed that the VEGFR3 inhibitor T72924 significantly inhibited the proliferation and tube-forming ability of lymphoendothelial cells, and its effect was superior to that of the VEGFR3 inhibitor MAZ51.

Claims

1. Use of selective VEGFR3 inhibitors and antiPD-L1 antibodies or their antigen-binding fragments in the treatment of lung cancer.

2. The use as described in claim 1, wherein, The lung cancer in question is non-small cell lung cancer.

3. The use as described in claim 1 or 2, wherein, The selective VEGFR3 inhibitor is T72924.

4. The use according to any one of claims 1-3, wherein, The anti-PD-L1 antibody is selected from B7-H1, atezolizumab, durvalumab, avelumab, envafolimab, or sugemalimab.

5. The use according to any one of claims 1-4, wherein, The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, or diabody.

6. A combination of drugs comprising a selective VEGFR3 inhibitor and an anti-PD-L1 antibody or its antigen-binding fragment.

7. The pharmaceutical combination of claim 6, wherein, The selective VEGFR3 inhibitor is T72924.

8. The pharmaceutical combination of claim 6 or 7, wherein, The anti-PD-L1 antibody is selected from B7-H1, atezolizumab, durvalumab, avelumab, envafolimab, or sugemalimab.

9. The pharmaceutical combination according to any one of claims 6-8, wherein, The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, or diabody.

10. The pharmaceutical combination of any one of claims 6-9, wherein the selective VEGFR3 inhibitor and the anti-PD-L1 antibody or their antigen-binding fragment are in separate or identical dosage forms.

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